Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to 98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.     

In vitro expression of partial resistance toPhytophthora palmivora by shoot cultures of papaya

Shoot apices ofCarica papaya were multiplied in vitro on solidified nutrient media supplemented with -naphthyl-acetic acid and 6-benzylaminopurine. The micropropagated shoots were inoculated in vitro, through a stem wound, with a sporangial suspension (1.2×10⁴ sporangia ml^-1) ofPhytophthora palmivora. The symptoms exhibited by the shoots in vitro were similar to those described previously for infection of the whole plant in the field. The time taken for the host tissue to become brown and to wilt and the time to sporulation of the pathogen were all recorded for each shoot of four varieties of papaya challenged with each of ten isolates ofP. palmivora. Significant differences were observed between host-pathogen combinations for these variables and host-specificity was detected amongst the isolates ofP. palmivora. The time taken for the shoot to wilt was positively correlated with the time to sporulation of the isolated but both these variables were negatively correlated with the time to browning of the shoot. In vitro selection for disease resistance will be useful during breeding programmes involving papaya genotypes which are maintained through clonal propagation.     

Effect of different carbon and nitrogen sources on the growth and sporulation of Claviceps microcephal (Wallr) TUL.

The effect of different carbon and nitrogen compounds on growth and sporulation ofC. microcephala (Wallr.)Tul. causing ergot disease of Bajra has been studied. Nine different sources of carbon were used but cane sugar was found to be the best source for both, growth and sportulation of the fungus. Glucose, sucrose and maltose gave good growth but fair sporulation. Lactose and sorbitaol proved to be the poor sources. However, fungus failed to utilize starch, dextrin and mannitol.Nineteen nitrogen compounds were tried for the growth and sporulation of the fungus. Best growth and sporulation were supported by peptone and glycine. L-asparagine, DL-valine, Urea, magnesium nitrate and L-proline supported good growth and fair sporulation except DL-valine where it was excellent. Poor growth was obtained on L-isoleucin, ammonium sulphate, potassium nitrate,-alanine, ammonium chloride, DL-aspartic acid and DL-methionine. Fungus failed to utilize thio-urea.     

Antibiotic-induced changes of mycelial growth ofBotrytis cinerea

The effect of antibiotics and metabolic inhibitors on mycelial growth ofBotrytis cinerea was followed. Inhibitors of protein synthesis, chloramphenicol, erythromycin and tetracycline inhibit growth or sporulation ofBotrytis cinerea. Ethidium bromide, 5-fluorouracil, phenylethylalcohol and K 20 cause granulation, vacuolization and undulation of hyphae. 2,4-Dinitrophenol, boromycin, macrotetrolides, monensin, scopathricin and TX₂ at subfungistatic concentrations induce intensive branching of hyphal tipsi.e. at the site of synthesis of the cell wall. In older hyphae grown in the absence of the antibiotics the branching begins after their addition, particularly in the septum region. When comparing the results referred to here with those obtained previously and on the basis of literature data it may be assumed that the changes in polarity of growth ofBotrytis cinerea might be caused primarily or secondarily by impairing membrane functions and formation of cell walls.     

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.     

Influence of nitrogen nutritions on asexual reproduction of five species Sphaeropsis sacc. in culture

Effect of fourteen different nitrogen sources on five species ofSphaeropsis i. e.S. cactinae, S. cycadis, S. jodhpurensis, S. muehlenbeckiae andS. punicae, obtained respectively from the infected parts ofCitrus medica, Cycas circinalis, Cordia gharaf, Muehlenbeckia platycladoes andPunica granatum was studied, under cultural conditions on Asthana and Hawker's Medium A by substituting its nitrogen with equal quantity of an other nitrogen source. All the present species reproduced well on potassium nitrate, sodium nitrate, ammonium sulphate and ammonium nitrate. None of the present species could grow on sodium nitrite. Out of all amino acids, no one was found to be excellent for the growth and sporulation of the present fungi. Most of the amino acids supported poor growth and sporulation of three of the five species ofSphaeropsis, S. cactinae, S. cycadis andS. punicae on L-asparagine, Glycine, L-cystine and L-alanine. Maximum and minimum pH for growth and sporulation ranged from 6.5 to 7.8 and 5.3 to 4.2 respectively. Decrease in adjusted pH was also observed for all the species on L-asparagine and DL-valine.     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.     

Factors modulating differentiation of spores: Characterization of mutants defective in sporulation in the cyanobacterium Anabaena doliolum (AdS strain)

Summary Mutants of Anabaena doliolum (AdS strain) altered with respect to the time of initiation and degree of sporulation were isolated following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine and hydroxylamine. The non-sporulating mutant showed a high phycocyanin (Pc): chlorophyll a (chl a) ratio (ca. 7.2) as compared to sporulating strains (Pc:chl a, 4.7–5.3). Also this strain seemed to have higher RNA pools per unit of genomic material as reflected in a higher RNA:DNA ratio. The data suggest that degradaton of phycocyanin and controlled RNA synthesis are prerequisites for sporulation. Mutants exhibiting non-sporulation and delayed initiation of sporulation accumulated more nitrogen through nitrogen fixation, probably indicating nitrogenase function over an extended vegetative phase.     

Catabolite-Induced Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

In response to nutrient limitations, Bacillus subtilis cells undergo a series of morphological and genetic changes that culminate in the formation of endospores. Conversely, excess catabolites inhibit sporulation. It has been demonstrated previously that excess catabolites caused a decrease in culture medium pH in a process that required functional AbrB. Culture medium acidification was also shown to inhibit Ï^H-dependent sporulation gene expression. The studies reported here investigate the effects of AbrB-mediated pH sensing on B. subtilis developmental competence. We have found that neither addition of a pH stabilizer, MOPS (pH 7.5), nor null mutations in abrB blocked catabolite repression of sporulation. Moreover, catabolite-induced culture medium acidification was observed in cultures of catabolite-resistant sporulation mutants, crsA47, rvtA11, and hpr-16, despite their efficient sporulation. These results suggest that AbrB-mediated pH sensing is not the only mechanism regulating catabolite repression of sporulation. The AbrB pathway may function to channel cells toward genetic competence, as opposed to other postexponential differentiation pathways.     

Naturally produced isocoumarins: inhibitors of calmodulinsensitive cyclic guanosine 3′,5′-monophosphate phosphodiesterase

Summary Three isocoumarins have been isolated from a strain ofStreptoverticillium sp. and all inhibit the calmodulin-sensitive cyclic guanosine 3,5-monophosphate phosphodiesterase (EC 3.1.4.17, Boehringer Mannheim). Two of the compounds, 6,8-dihydroxy-7-methoxy-3-methyl isocoumarin and 6,7,8-trihydroxy-3-methyl isocoumarin have previously been isolated fromStreptomyces. The third fermentation product, 6,8-dihydroxy-3-methyl isocoumarin, was also found as a metabolite ofCeratocystis minor, a fungal species associated with the blue stain disease of pine [2,3].     

Sporulation of auxotrophic mutants ofBacillus subtilis in suboptimal concentrations of essential amino acids

The effect of the amino acid concentration on sporulation of five mutants ofBacillus subtilis requiring histidine, isoleucine, leucine, lysine and methionine for growth was studied. Low amino acid concentrations, permitting about 10–20% of maximum growth, inhibit the ability of the bacteria to sporulate on completing vegetative growth. In medium concentrations (from 25% of maximum growth), leucine, isoleucine, histidine and methionine induce sporulation, while the concentration of the other nutrients in the medium is still adequate for further vegetative growth. The percentage of sporulation depends on the amino acid concentration and is characteristic for each amino acid. The lysine mutant strain 1399 was not capable of sporulation unless cultivated in the presence of the lysine concentration optimal for growth. The effect of amino acid deficiency depends on the cultivation method.     

Effect of glucose on sporulation and extracellular amylase production byClostridium perfringens type A in a defined medium

The effect of glucose and other sugars on sporulation and extracellular amylase production byClostridium perfringens NCTC 8679 type A in a defined medium was studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity, while disaccharides such as lactose, maltose, and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10mm except for fructose (25mm). Levels of heat-resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in this strain ofC. perfringens.     

Two strains ofVibrio cholerae non-O1 possessing somatic (O) antigen factors in common withV. cholerae serogroup O139 synonym “Bengal”

Two strains (O22 reference strain, 169–68, and strain 490–93 isolated from a patient with diarrhea in Thailand) ofVibrio cholerae non-O1 possessing somatic (O) antigen factors in common withV. cholerae O139 synonym Bengal are described. The O antigens of these two strains were closely related to that ofV. cholerae O139 in an a,b-a,c type of relationship, but were not completely identical with serogroup O139. Therefore, both these strains are not classified into the O139 serogroup ofV. cholerae, because they have their own major antigens. As the strain 490–93 could not be placed into any of the 154 established O serogroups ofV. cholerae, this strain was assigned to a new serogroup, O155. For practical use, the diagnostic antiserum prepared against the O139 reference strain (MO45, ATCC 51394) ofV. cholerae must be absorbed with reference strains 169–68 and 490–93 representing serogroups O22 and O155 ofV. cholerae to remove cross-reacting agglutinins of the O22 and O155 strains, respectively.     

Effect of Inhibitors of Bacterial Cell Wall Synthesis on the Growth of Anabaena variabilis,a Blue-green Alga

The effects of penicillins and several other antibiotics (vancomycin, ristocetin, bacitracin, novobiocin, and d-cycloserine), all known as inhibitors of bacterial cell wall synthesis, were tested on the growth of Anabaena variabilis, a strain of blue-green alga.

All the antibiotics tested inhibited the growth of Anabaena variabilis at concentrations ranging from 10 µg to 1 mg per ml. However, penicillins and the other antibiotics tested did not inhibit growth of strains of green algae.     

Natural resistance of the mycelial culture of the mushroom,Panaeolus papillonaceus,towards growth inhibition by polyene antibiotics

The mycelial culture of the mushroomPanaeolus papillonaceus showed high tolerance (150 g/ml) of polyene antibiotics (nystatin, amphotercinin B) present in the growth medium and protoplast of the fungus regenerated normally in the presence of the antibiotics. Both antibiotics inhibited growth of other mushroom strains at concentrations from 10 g/ml to 20 g/ml. Because polyene antibiotics interact with free membrane sterol of the sensitive fungi, the sterol present in the mycelia ofP. papillonaceus was studied. Extraction of sterol from the mushroomP. papillonaceus required primary treatment of the dried mycelia with alkali, and only ergosterol was identified as present as the extracted sterol. No sterol or sterol conjugate (fatty acid ester) could be extracted directly from the mycelia by petroleum ether, chloroform, or methanol without prior alkali treatment. Homogenization of the mycelia and subsequent treatment of the homogenate with detergent or chaotropic ions did not release any sterol conjugate in the aqueous phase. The unique nature of the sterol component present in the mycelia ofP. papillonaceus was indicated.     

Galactose metabolism inErwinia carotovora Subsp.carotovora

The pyrimidine analogue 5-fluorouracil was shown to be a potent inhibitor of the growth ofMethanobacterium thermoautotrophicum strain Marburg (50% inhibition of growth at 1 g ml^–1). The nucleoside, 5-fluorodeoxyuridine, also inhibited growth, but the nucleotide 5-fluorodeoxyuridylate did not inhibit, nor did 5-fluorocytosine. Several nucleobases and nucleosides were used as potential antagonists of fluorouracil and fluorodeoxyuridine. Of these, only uracil in excess over fluorouracil relieved the inhibition of growth. These results imply that a pyrimidine salvage pathway is present inM. thermoautotrophicum. 5-Fluorouracil does not inhibit methane production. Although treated cultures produced less methane than did controls, more than twice as much methane was synthesized per cell. This result suggests that methanogenesis is uncoupled from growth by 5-fluorouracil.     

Beziehungen zwischen katabolischer Repression und Sporulation bei Bacillus subtilis

Acetoin dehydrogenase can be catabolite repressed by numerous sources of carbon. The following results point out that the catabolite repression of this enzyme and the inhibition of sporulation are mediated by the same mechanism:

1. Mutants, able to synthesize acetoin dehydrogenase in the presence of glucose, sporulate in glucose medium at a higher rate than the standard strain.
2. The catabolite repressing effect of a compound and its ability to inhibit sporulation are in a direct relation to each other.
3. The limitation of inorganic phosphate in the growth medium, which is known to favour sporulation, counteracts the catabolite repressing effect of glucose.

     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

AbrB and Spo0E Control the Proper Timing of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

We have shown previously that Spo0AP-dependent sinIR operon expression was substantially down-regulated in abrB null mutant backgrounds. In this report, we show that loss of function mutations in abrB also cause phosphorelay gene expression to be down regulated. abrB null mutations caused diminished vegetative growth-associated sporulation and resulted in a significant reduction in sporulation frequencies at T₂₄. These mutants, however, sporulated at wild-type levels at T₄₈, indicating that sporulation timing was affected. The rvtA11 mutation in spo0A, a deletion mutation in spo0E, and a null mutation in hpr (scoC) rescued sporulation and Spo0AP-dependent gene expression in an abrB mutant background. These data indicate that AbrB and Spo0E may comprise a checkpoint system that regulates the progression of sporulation, allowing exploration of alternate cell states prior to the irrevocable commitment to sporulation.