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J. Biosci., Vol. 2, Number 4, December 1980. pp. 321–328 © Printed in India Spore and crystal formation in Bacillus thuringiensis var. thuringiensis during growth in cystine and cysteine. S. RAJALAKSHMI and Y. I. SHETHNA Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore 560012, MS received 24 July 1980; revised 22 October 1980. Abstract. The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation i
   J. Biosci., Vol. 2, Number 4, December 1980. pp. 321–328 ©   Printed in India Spore and crystal formation in  Bacillus thuringiensis var.  thuringiensis during growth in cystine and cysteine. S. RAJALAKSHMI and Y. I. SHETHNA Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore 560012,   MS received 24 July 1980; revised 22 October 1980. Abstract. The effect of the addition of different concentratons of cystine and cysteine onsporulation and parasporal crystal formation in  Bacillus thuringiensis var. thuringiensis wasstudied. The effect was well pronounced when the cystine/cysteine additions were made afterthe stationary phase. Heat stable spores and crystals were formed when the culture was providedwith a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentrationof cystine or cysteine (0.15%), only heat labile spores were formed without the production of thecrystal. When the cystine/cysteine concentration was high (0.25%), spore and crystal formationwere completely inhibited. Partial reversal of inhibition of sporulation was brought about bysodium sulphate or zinc sulphate and lead, copper, cadmium or cobalt acetate at 0.2 mM or at0.2% of sodium or potassium pyruvate, citrate, cisaconitate, oxalosuccinate, ∝ -keto-glutarate,succinate, fumarate, malate, or oxalacetate. Glutamate (0.2%) overcame the inhibitory effect of cystine/cysteine completely. The structural changes observed using phase contrast microscopywere dependent upon the concentration of cystine/cysteine. Keywords.  Bacillus thuringiensis var. thuringiensis. cystine, cysteine, spore, crystal, reversal of inhibition. Introduction    Bacillus thuringiensis, an important insecticide (Bulla et al., 1975) is toxic mainly tolepidopteran larvae (Rogoff and Yousten, 1969). The toxicity resides in an intracellularparasporal crystalline inclusion (Angus, 1954,1956a, 1956b, 1956c; Rogoff and Yousten,1969; Heirnpel and Angus 1960; Nickerson et al., 1974). Upon ingestion of theproteinaceous crystal by susceptible larvae, proteolytic enzymes in the larval gut juice hydrolyze the crystal protein into as yet undefined toxic moieties. This isfollowed by paralysis and eventual death of the larvae (Angus, 1954; Angus, 1956a;Angus and Heimpel, 1959).Since sporulation and germination in bacilli are dependent on the nutritionalstatus of the organism (Hardwick and Foster, 1952), a study of the nutritional require-ment of   Bacillus thuringiensis var. thuringiensis is important for delineating thecontrol mechanisms which regulate spore and parasporal crystal formation. Certainamino acids support growth, sporulation and crystal formation of   B. thuringiensis   Abbreviation: cys/cysSH–cystine and cysteine 321  322  Rajalakshmi and Shethna var. thuringiensis, while others inhibit the growth (Singer  et al., 1966; Singer andRogoff, 1968; Bulla et al., 1975; Nickerson and Bulla, 1975; Rajalakshmi and Shethna,1977). A lower concentration of cystine (Nickerson and Bulla, 1975) or cysteine(Rajalakshmi and Shethna, 1977) promotes growth, sporulation and crystal formationin  Β . thuringiensis, while at a higher concentration of cys/cysSH, only the vegetativegrowth was observed, (Rajalakshmi and Shethna, 1977).We report here a detailed study of the effect of various concentrations as well asthe time of addition of cys/cysSH on spore and crystal formation in  Β . thuringiensis var. thuringiensis and the reversal of inhibition of sporulation by various compounds.   Materials and methods Organism and culture conditions Bacillus thuringiensis Serotype I was obtained from Professor  Η . de Barjac, InstitutePasteur, Paris, France. The culture was maintained on nutrient agar slants. Theminimal medium and the inoculum were prepared as described earlier (Rajalakshmiand Shethna, 1977).  Effect of cys/cysSH  To study the effect of various concentrations of cys/cysSH the minimal mediumcontaining 1% glucose was supplemented with 0.05, 0.1, 0.15, 0.2 and 0.25% of cys/cysSH, respectively. Growth, sporulation and crystal formation were monitoredfrom time to time.  Effect of addition of cys /cysSH after the stationary phase In another set of experiments,  B. thuringiensis var. thuringiensis cells were grown ina minimal medium containing 1.0% glucose and 0.05% cys/cysSH. Different concentra-tions of cys/cysSH (i.e. 0.05, 0.1, 0.15 and 0.2%) were added at 0, 1, 2,3 and 4 h after the stationary phase was reached and their effects on spore and crystal formationwere studied. Microscopic studies For phase contrast microscopy, control and experimental cells were harvested at themid log phase and 10 h after the attainment of the stationary phase. The cells werewashed and suspended in 0.1 Μ potassium phosphate buffer pH 7.2. A wet smear of the suspension was observed immediately under a light microscope (Zeiss photomicroscope, W. Germany).  Reversal of inhibition of sporulation A reversal of inhibition of sporulation, caused by cys/cysSH was effected by theaddition of divalent cations, intermediates of the tricarboxylic acid cycle or glutamateindividually. Sodium or zinc sulphate and lead, copper, cadmium and cobalt acetatewere used (2 mM). Sodium or potassium puruvate, citrate, cis -aconitate, oxalosucci-nate ∝ -keto-glutarate, succinate, fumarate, malate, oxalacetate and glutamate wereused at a concentration of 0.2% after filter sterilization.Additions were made 60 to 90 min prior to the stationary phase, when varyingconcentration of cys/cysSH was provided in the growth medium. Whereas, when  Spore-crystal  formation in Bacillus thuringiensis 323 the cys/cysSH additions were made after the stationary phase, the substances wereadded 10 to 15 min prior to the addition of excess cys/cysSH.Cys and cysSH, wherever mentioned, were used individually. Growth was moni-tored by measuring the absorbance at 600 nm in a Bausch and Lomb Spectronic-20colorimeter. Sporulation and crystal formation were monitored by the observationof a wet smear of the culture under a phase contrast microscope (Zeiss dark phasecontrast microscope). Heat-stable spores were estimated by a standard procedure(Nickerson and Bulla, 1975). When the cells were grown with low concentration of cys/cysSH (0.05%), heat-stable spores and crystals were produced; under such acondition, there was no heat-labile spore formation. When the cells were providedwith a moderate concentration of cys/cysSH (0.15%), only heat labile spores wereproduced. The heat-labile spores were found to be susceptible to lysozyme treat-ment, whereas the heat-stable spores were not succeeptible to such treatment. Theheat-labile spores were estimated by plating on nutrient agar plates after appropriatedilution. Heat lability and stability were confirmed by lysozyme treatment (Stahly et al., 1978). Cells grown 0.05% cys/cysSH and 1 % glucose were taken as the controlfor all the studies given under Materials and Methods. Toxicity of the crude celllysate was checked by feeding the pellet obtained after centrifugation of the crudecell lysate at 8,000 g for 10 min, to the second or third instar larvae of silkworm moth (Bombyx mori). The crude pellet was suspended in distilled water, sprayed onmulberry leaf (1 mg protein) and fed to 20 larvae. Chemicals Intermediates of tricarboxylic acid cycle and glutamate were obtained from SigmaChemical Co., St. Louis, Missouri, USA. L-Cystine, L-cysteine* and all the salts of divalent cations were obtained from E. Merck, Darmstadt, Germany. All the otherchemicals were of Analar or Reagent grade. Results Spore and crystalformation in relation to cys/cysSH concentration The efficiency of spore and crystal formation was studied in relation to cys/cysSHconcentration. It was noticed that in the control, as well as in the presence of cys/cysSH (0.05%), the efficiency of spore and crystal formation was 100 percent. Ata concentration of 0.1% of cys/cysSH, the efficiency was drastically reduced. At0.15 and 0.2% of cys/cysSH, only heat-labile spores were formed with completeinhibition of crystal formation. At 0.25% cys/cysSH, there was complete inhibitionof spore and crystal formation in  B. thuringiensis var. thuringiensis (figure 1). * Cysteine was freely soluble upto 0.112g/1 at25 °C. Whenever needed, it was solubilized with acid(0.1NHCl) and then added to the medium. The pH of the medium was adjusted to 7.2 after the cys/cysSHaddition. (Reference: The Merck index of chemicals and drugs, 7th ed. Merck and Co. inc., Rahway,New Jersey, USA 1960.)  324  Rajalakshmi and Shethna   Figure 1.  Effect of cys/cysSH on spore and crystal f  ο rmation in  B. thuringiensis var. thuringiensis. Heat-labile and heat-stable spores were calculated as given under Materials and Methods. Toxicitywas checked by feeding to 2nd or 3rd instar silkworm (Bombyx mori) as mentioned in the text.SC-Heat stable spores and crystals. HLS-Heat labile spores. –SC: No spore and crystal.Since the absorbance increased with respect to cys/cysSH concentration, all the samples werediluted so as to obtain an absorbance equal to control (0.05 per cent cys/cysSH). After dilution,the number of spores per ml was estimated. Cystine, ( ■ );Cysteine, ( □ ).  Effect of addition of cys/cysSHat the stationary phase It was noticed that when 0.05% of cys/cysSH was added at 0 h of the stationary phase,heat-stable spores and toxic cystals were formed. At0.1 and 0.15%, only heat-labilespores were produced with no crystal formation. Where as, when 0.2% of cys/cysSHwas added, spore and crystals were not formed (table 1) microscopically, and the bio-assay also showed no toxicity.  Effect of addition of cys/cysSH after the stationary phase Sporulation and parasporal crystal formation were found to be inhibited even at theconcentration of 0.15% of cys/cysSH when the addition was made 1 h after the onsetof the stationary phase (table 1). The efficiency of sporulation decreased as the timeof addition of excess cys/cysSH was increased.
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