Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Climatic factors influencing spore production in Alternaria brassicae and Alternaria brassicicola

Sporulation in A. brassicae and A. brassicicola on naturally-infected leaf discs of oilseed rape and cabbage required humidities equal to or higher than 91.5% and 87% r.h. respectively. The optimum temperatures for sporulation were 18–24°C for A. brassicae and 20–30°C for A. brassicicola at which temperatures both fungi produced spores in 12–14 h. Above 24°C sporulation in A. brassicae was inhibited. At sub-optimal temperatures sporulation times for A. brassicicola were significantly longer than for A. brassicae with the differences increasing with decrease in temperature. Interrupting a 16-h wet period at 20°C with a period of 2 h at 70% or 80% r.h. did not affect sporulation in either fungus but a dry interruption of 3–4 h inhibited sporulation in both. Exposure of both fungi to alternating wet (18 h at 100% r.h., 20°C) and dry periods (6 or 30 h at 5565% r.h., 20°C) did not affect the concentration of spores produced in each wet period. Sporulation times were not affected by either the host type of the age of the host tissue. White light (136 W/m²) inhibited sporulation in A. brassicae with the degree of inhibition increasing with increasing light intensity. The effect of light on sporulation in A. brassicicola was not tested.     

Selectivity in Protein Degradation during Sporulation of Bacillus subtilis

The breakdown of cellular protein was investigated in Bacillus subtilis labeled with glycine-2-³H or L-phenylalanine-U-¹⁴C at different stages of vegetative growth and sporulation. In cells labeled with l-phenylalanine-U-¹⁴C, multiple protein turnover was observed. However, in cells labeled with glycine-2-³H, the patterns of protein turnover were quite different in the stages of growth and sporulation; proteins which were labeled at the early stationary phase were degraded rapidly, but those labeled at the late sporulation stage were hardly degraded. It was found that glycine incorporated into cells at the late sporulation stage was mainly utilized for biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amounts of glycine is little subject to further degradation.     

Effect of culture conditions on growth and sporulation ofBacillus thuringiensis subsp.israelensis and development of media for production of the protein crystal endotoxin

Summary The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 10⁹/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.     

Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse-labeling experiments indicated that [¹⁴C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t₅ (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [¹⁴C]phenylalanine was observed when the isolated forespores at t₅ were incubated with the corresponding mother-cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t₅ was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the ¹⁴C-labeled cytoplasmic protein had a molecular weight of about 20,000, and that a protein having the same molecular weight was present in the t₅ forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the ¹⁴C-labeled membranous-soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.     

In Vivo and In Vitro Function of the Intracellular Proteolytic Apparatus in Nongrowing Bacillus megaterium Under Heat Stress

In Bacillus megaterium sporulating at 35°C, up to 90% of 10-min pulse-labeled proteins were degraded. Degradation proceeded in two waves. Short-lived proteins, i.e., intrinsically labile proteins and proteins made short-lived because of starvation, were mostly degraded during the reversible sporulation phase. Their amount corresponded to 20% or slightly more during 2 h. The second wave of protein degradation, which followed during the irreversible sporulation phase at 35°C, increased the amount of total degradable pulse-labeled proteins to about 90%. This wave was absent in the isogenic asporogenic mutant 27-36 or in the wild strain, whose sporulation was inhibited by increased temperature. The proportion of degradable proteins was thus reduced to less than 40% in the asporogenic mutant incubated at 35°C and to 46% in the wild strain whose sporulation was suppressed by the temperature of 47°C. Unlike sporulating cells, these cells were thus capable of degrading short-lived and denatured proteins, but were not able to degrade most of other proteins. The in vitro protein degradation was substantially enhanced by increasing the Ca²⁺ concentration, suggesting a role of Ca²⁺-dependent proteinase(s) in the process. Received: 23 July 1998 / Accepted: 19 August 1998     

Effects of Indole-3-acetic Acid and Gibberellin on Synchronous Cultures of Chlorella fusca

The applicability of synchronous cultures of Chlorella fusca as a reproducible experimental system for the study of growth regulators has been investigated using indole-3-acetic acid (IAA) and gibberellin.

• 1 None of these compounds stimulated growth or sporulation.
• 2 IAA inhibited growth and sporulation at concentrations higher than 6 × 10^?5M, the effect increasing with increasing acidity. Gibberellin had no effect.

     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168

Pleiotropic mutations of the chromosome of Bacillus subtilis 168 affecting simultaneously the levels of extracellular levansucrase and proteolytic activities are described. These mutations have been mapped at the sacU locus identified by PBS 1 mediated transduction. Several pleotropic hyperproducers and pleiotropic hypoproducers of these extracellular enzymatic activities, genotypically designated sacU^h and sacU⁻ respectively, have been isolated. sacU^h mutants are capable of sporulation in rich media or in mineral media containing amino acids in the presence of an excess of glucose in both cases; under these conditions the sporulation of the wild type strain 168 is inhibited. One pleiotropic mutation conferring hyperproduction of levansucrase and proteolytic activities was mapped at the sacQ locus distant from sacU.The sacU and sacQ mutants may be affected in a not yet identified regulation mechanism which controls simultaneously the production of several extracellular enzymatic activities and the sporulation conditions of B. subtilis 168.     

Turnover kinetics of proteins labeled in different sporulation phases ofBacillus megaterium

Bacillus megaterium was labeled by 10-min pulses of¹⁴C-leucine at the end of the growth phase or at 1, 3.5 and 5 h after transfer to a sporulation medium. Proteins labeled during growth or reversible sporulation phase were degraded in two-phase kinetics,i.e. a decreasing degradation rate was followed by its substantial increase. Proteins labeled during the irreversible sporulation phase were degraded at a continuously decreasing degradation rate only. However, when the amount of degraded proteins was expressed as a portion of proteins degradable during the whole sporulation cycle, the degradation was rapid and followed similar kinetics irrespective of the time of labeling. The degradation constants fluctuated in this case between 0.207/h and 0.275/h. The protein fraction insensitive to turnover increased with the time of incubation in the sporulation medium in parallel to the amount of proteins appearing in spores.     

Improved production of erythromycin A by expression of a heterologous gene encoding S-adenosylmethionine synthetase

An S-adenosylmethionine synthetase (SAM-s) gene from Streptomyces spectabilis was integrated along with vector DNA into the chromosome of a Saccharopolyspora erythraea E2. Elevated production of SAM was observed in the recombinant strain Saccharopolyspora erythraea E1. The results from the bioassay showed that the titer of erythromycin was increased from 920 IU ml⁻¹ by E₂ to approximately 2,000 IU ml⁻¹ by E₁. High performance liquid chromatography (HPLC) analysis revealed that there was a 132% increase in erythromycin A compared with the original strain, while the erythromycin B, the main impurity component in erythromycin, was decreased by 30%. The sporulation process was inhibited, while the SAM-s gene was expressed. The addition of the exogenous SAM also inhibited sporulation and promoted an increase in erythromycin titers. An erratum to this article can be found at     

Effect of pH on Adenine and Amino Acid Uptake During Sporulation in Saccharomyces cerevisiae

During the early stages of sporulation in Saccharomyces cerevisiae, the pH of the acetate sporulation medium rises to values of 8.0 or higher. Associated with this rise in pH is a reduced cell permeability to certain precursors of ribonucleic acid (RNA), deoxyribonucleic acid or protein. Uptake of adenine, alanine, and leucine was optimal at pH 5.6 to 6.0, but sporulation was inhibited when the sporulation medium was buffered below pH 7.0. Cellular impermeability can be largely overcome by adjusting the acetate sporulation medium to pH 6.0 for optimal uptake of ¹⁴ C-adenine during short pulses without any apparent effect on sporulation. Sporulating cells pulse-labeled 20 min at pH 6.0 incorporated 40 times more ¹⁴C-adenine into RNA than sporulating cells pulse-labeled at pH 8.0. This increased incorporation can be attributed to a 100-fold increase in labeled adenosine triphosphate in cells pulse-labeled at pH 6.0 where maximum uptake occurs.     

Effects of Exogenous Adenosine 5′-triphosphate 3′-diphosphate on Growth and Sporulation of Bacillus subtilis

Exogenous adenosine 5′-triphosphate 3′-diphosphate (pppApp) had interesting effects on the cell cycle of B. subtilis IFO 3027. The growth rate was reduced by the addition of 1 mm pppApp, and the vegetative cell form was significantly changed. Moreover, the sporulation frequency was increased by 100 times or more as compared with the culture without pppApp. The sporulation process seemed to be stimulated around t₀. pppGpp and ppGpp also showed the same effects as pppApp. Among these effects, depression in growth rate was restored by Mg²⁺ and Ca²⁺, and stimulation of sporulation was inhibited by Mg²⁺, Ca²⁺ and certain carbon sources, such as glucose and glycerol. On the other hand, casamino acids or monovalent cations showed no influence on the pppApp effects. pppApp was not incorporated into cells in experiments with radioactive pppApp.     

Relation between inhibition of Bacilli sporulation and synthesis of lytic enzymes.

In two strains of Bacillus, the synthesis of two specific lytic enzymes was studied concomitantly with an inhibition of the sporulation: LD-carboxypeptidase synthesis was unaffected whereas γ-D-glutamyl-(L)meso-diaminopimelyl endopeptidase synthesis was shown to be closely related to sporulation. The endopeptidase production is totaly inhibited when netropsin inhibits sporulation in B. sphaericus and is low in B. subtilis Thy^?A when sporulation is inhibited by thymidine starvation. This enzyme seems directly connected with the sporulation sequence.     

Biochemical and physiological properties of an extracellular protease produced by Bacillus megaterium

A protease, excreted by a sporogeneous strain of B. megaterium, growing exponentially in a minimum glucose ammonium medium, was isolated. It is a neutral endopeptidase, stabilized by Ca⁺⁺, inhibited by o-phenanthroline, but not by di-isopropylfluorophosphate. The specificity, studied on insulin B-chain, glucagon, cytochrome c, and dipeptides substrates, indicated the need for a dipeptide backbone with both substituted amino and carboxyl groups. A requirement was observed for a nonpolar lateral chain in the amino acid whose amino group was involved in the peptide bond (Leu, Phe, Ala, He, Val). Rates of hydrolysis varied also with the amino acid whose carboxyl group was involved (e.g., His > Ser > Ala > Gly). In complex medium, supplemented with Yeast Extract, the biosynthesis of the protease was repressed during growth, but the same enzyme was excreted during sporulation. The repression was apparently of the same nature as that controlling sporulation during and after growth (e.g., repression by a mixture of amino acids or high concentration of glucose). An asporogeneous mutant showed a normal product ion of protease under all conditions, and a low intracellular protease turnover after growth. A mutant unable to produce protease showed a normal sporulation and a high protein turnover. This protease, here termed megapeptidase, seems to be a typical growth enzyme, not related to either the sporulation process or to the protein turnover after growth.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.     

Effects on growth and sporulation of inactivation of a Bacillus subtilis gene (ctc) transcribed in vitro by minor vegetative cell RNA polymerases (E-sigma 37, E-sigma 32)

Summary The E-³⁷ gene ctc was inactivated by a site-specific insertion into the Bacillus subtilis chromosome. The resulting mutation inhibited sporulation by 95% at elevated temperatures (48° C). If the ctc ^- mutation is placed in a strain that carries a mutation in the closely linked but distinct spoVC gene, ctc now affects both growth and sporulation at elevated temperatures. Growth of the ctc ^- spoVC285 strain was transiently inhibited when exponentially growing cultures were shifted from 37° C to 48° C. A similar, but less pronounced growth lag, was also seen in a B. subtilis strain carrying only the spoVC-285 mutation. This finding suggests that both the ctc and spoVC products function in vegetatively growing B. subtilis.