Research in Microbiology 154 (2003) 49–53 www.elsevier.com/locate/resmic
Nutritional and cultural parameters influencing antidipteran delta-endotoxin production Melek Özkan a , Filiz B. Dilek b , Ülkü Yetis b , Gülay Özcengiz a,∗ a Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey b Department of Environmental Engineering, Middle East Technical University, 06531 Ankara, Turkey
Received 5 September 2002; accepted 22 October 2002 First published online 25 October 2002
Abstract In this study, various nutritional and cultural parameters influencing diptera-specific delta-endotoxin synthesis by Bacillus thuringiensis subsp. israelensis HD500 were investigated. Of various inorganic nitrogen sources, the highest yields of Cry11Aa and Cry4Ba proteins were obtained on (NH4 )2 HPO4 . Among carbon sources, inulin, dextrin, maltose, lactose, sucrose, whey and glycerol were all stimulatory, while glucose, starch and molasses were suppressive. High concentrations of inorganic phosphate (50 to 100 mM K2 HPO4 ) were required for an effective synthesis of Cry4Ba. Mn was the most critical element for the biosynthesis of both toxins at 10−6 M concentration. Mg and Ca favored production when provided at 8 × 10−3 M and 5.5 × 10−4 M concentrations, respectively, while Fe, Zn and Cu negatively influenced biosynthesis. Cry4-toxin synthesis was best at neutral pH and also when the organism was grown at 25 ◦ C. Throughout the study, the extent of growth and sporulation of the producer organism was also monitored. 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Delta endotoxin; Regulation; Bacillus thuringiensis subsp. israelensis
1. Introduction The spore-forming bacterium Bacillus thuringiensis produces intracellular inclusions which are highly effective and specific toxins having great potential in agriculture and for the control of disease-related insect vectors. Inclusions ingested by larvae are solubilized and converted to active toxins in the midgut [1,3]. Bacillus thuringiensis serovar israelensis has been used since the 80’s in several programs for controlling blackfly and mosquito species worldwide. It is considered highly toxic to the target organisms and environmentally safe. Furthermore, no resistance has been detected in populations submitted to long-term exposure to this pathogen [16]. The crystals of B. thuringiensis subsp. israelensis contain four major endotoxins, designated Cry4A (125 kDa), Cry4B (134 kDa), Cry11A (67 kDa), and Cyt1Aa (27 kDa). No single crystal protein is as toxic as the intact crystal complex [6,10]. These protein toxins are expressed concomitant with sporulation. Environmental factors play a critical role in * Correspondence and reprints.
E-mail address:
[email protected] (G. Özcengiz).
modulating the differentiation pattern and synthesis of toxins which form a distinct group of secondary metabolites; thus their synthesis has considerably narrower tolerances for concentrations of specific trace metals and inorganic phosphate, as well as for ranges of temperature, pH and redox potential, than does growth of the producer cells [3,20]. Also, the repression or inhibition of secondary metabolism by catabolism of rapidly utilized carbon sources, especially glucose (carbon catabolite regulation) and nitrogen sources, especially ammonium, (nitrogen catabolite regulation) has been frequently reported and well illustrated [8]. In general, conditions for the culture of B. thuringiensis are optimized to achieve both high cell densities and high sporulation rates. In this attempt, a careful balance of substrates must be provided to avoid high cell densities with little or no sporulation [4]. Nevertheless, it has also been reported that a high spore count is not sufficient to ensure good toxicity [17]. Our preliminary studies involving protein profile analyses of sporulated cultures of three different B. thuringiensis subsp. israelensis strains (HD500, 4Q2-72 and HD14) revealed that Cry4Ba, Cry11Aa and Cyt1A toxin components were common to all of the strains while Cry4A and Cry4C proteins could not be detectable in any of them. In the
0923-2508/02/$ – see front matter 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 2 3 - 2 5 0 8 ( 0 2 ) 0 0 0 0 6 - 2
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present study, the effects of various nutritional and cultural factors on the biosynthesis of Cry4Ba and Cry11Aa toxins in B. thuringiensis subsp. israelensis HD 500 were determined and compared to their effects on growth and sporulation of the organism.
2. Materials and methods 2.1. Strains and media B. thuringiensis subsp. israelensis HD500 was kindly provided by Prof. Dr. Zeigler, Bacillus Genetic Stock Center, The Ohio State University, USA. Yousten’s medium (YSM) was used for cultivation of the strain. The medium contains (g l−1 in distilled water) glucose, 1.0; (NH4 )2 SO4 , 2.0; K2 HPO4 , 0.5; yeast extract, 2.0; MgSO4 ·7H2 O, 0.2; CaCl2 ·2H2 O, 0.08; MnSO4 ·H2 O, 0.05, pH 7.3. The cultures were incubated by shaking at 30 ◦ C for 12–14 h. Samples were taken from the cultures at 2-h intervals and used for quantitative determination of growth which was measured spectrophotometrically as absorbance at 600 nm. Viable counts, spore counts and sporulation frequency (ratio of heat resistant spores to viable cells) were determined as described by Özcengiz and Alaeddino˘glu [14]. Extraction of spores with crystals and SDS-PAGE of proteins were made as described by Donovan et al. [9]. Silver staining of the gels was performed as in Blum et al. [5]. Gels were photographed by the Vilber Gel imaging system and the amounts of toxins were detected by the PD Quest Program (BioRad).
3. Results In order to study the effect of the inorganic nitrogen sources on toxin formation, (NH4 )2 SO4 which is found in YSM at a concentration of 2 g l−1 was replaced by an equal amount of a different inorganic nitrogen source. Results showed that Cry4Ba production and sporulation were poor when nitrate salts (ammonium or potassium nitrate) were used as the sole inorganic nitrogenous substrate. Among different salts of ammonium, (NH4 )2 HPO4 greatly stimulated Cry4Ba toxin synthesis and sporulation (Fig. 1). Growth occurred at nearly the same rate in all media having different inorganic nitrogen sources (data not shown) whereas sporulation frequency was the highest on NH4 Cl. The effects of organic nitrogen sources were tested by replacing (NH4 )2 SO4 with an equal amount (2 g l−1 ) of glutamate or urea. Alternatively, both (NH4 )2 SO4 and yeast extract were also replaced by equal amounts (4 g l−1 as a total) of peptone, soybean flour, cornsteep liquor or casamino acids. All of these replacements led to a significant decrease in toxin yields (data not shown). Various carbon sources (each at a final concentration of 1 g l−1 ) were next compared for their effects. Glucose,
Fig. 1. Effect of inorganic nitrogen sources on toxin production and sporulation.
starch and molasses were repressive and/or inhibitory for toxin production, blocking in particular the formation of the 134 kDa (Cry4Ba) component (Fig. 2). In contrast, dextrin, whey, maltose, lactose, inulin, glycerol and sucrose influenced the toxin biosynthesis in a positive manner. These sugars supported good growth as well. Sporulation was poor in maltose medium as compared to the others; with lactose or dextrin, on the other hand, the sporulation efficiency was maximal. Taken together, dextrin appears to be the carbon source of choice as it provides the maximum levels of Cry4Ba and Cry11Aa toxin proteins and sporulation. The rest of our study involved an assessment of the effects at different levels of various parameters (inorganic phosphate, minor elements/trace metals, pH and temperature) that are known to affect secondary metabolism. The optimum level for each parameter as well as the maximum toxin and spore yields were tabulated in Table 1. The synthesis of Cry4Ba protein was particularly responsive to the level of inorganic phosphate in that its formation required the provision of K2 HPO4 at concentrations significantly higher than that routinely included in the medium (2.8 mM). The highest yields of both Cry4Ba and Cry11Aa, and the highest sporulation frequency were obtained when the cells were grown on 50–100 mM K2 HPO4 . Mn level was also critical for effective synthesis of crystal toxin complements. 10−6 M Mn specifically favored biosynthesis. This was also the optimum concentration for sporulation of the producer organism. Growth remained almost the same except for a high concentration of 10−2 M which was toxic to the cells. Cry4Ba and Cry11Aa synthesis did not seem to be influenced by Mg in a concentration range of 0 to 4 × 10−4 M of this element. However, a slight stimulation of Cry4Ba production was detectable when the concentration was raised to 8 × 10−3 M. While sporulation was best on 4 × 10−4 M MgSO4 , growth was not markedly influenced by varying the Mg concentration. When the Ca effect was investigated, the maximum synthesis of both toxins was obtained with 5.5 × 10−4 M Ca which corresponds to the amount originally contained in YSM. On the other hand, optimum growth and endospore formation required a higher concentration of 5.5 × 10−3 M (data not shown). Unlike the above-mentioned
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Fig. 2. Effect of carbon sources on toxin production and sporulation. Table 1 Optimum conditions for delta-endotoxin production and respective sporulation frequencies Parameter/studied range of level Inorganic phosphate (1–250 mM) MnSO4 (0–10−2 M) MgSO4 (0–8 × 10−3 M) pHb (5.5–8.5) Temperature (25–42 ◦ C)
Optimum level 50–100 mM 10−6 M 8 × 10−3 M 7.0 25 ◦ C (for Cry4Ba) 30 ◦ C (for Cry11Aa)
Amount of toxina (µg ml) Cry4Ba
Cry11Aa
2.6 12 3 2.5 1.7 0.3
6.5 12 6.2 3 2.1 4
Sporulation frequency (S/V) 1.2 × 10−1 4.0 × 10−1 3.5 × 10−2 1.0 7.6 × 10−2 5.7 × 10−3
a Cry4Ba and Cry11Aa toxin yields in control media were almost invariable, around 0.2 µg ml and 3 µg ml, respectively, in different sets of experiments. b In buffered media.
minerals, Zn, Cu and Fe negatively influenced toxin production (data not shown). Inclusion of Zn and Cu, especially at concentrations higher than 10−7 M, also inhibited growth and sporulation. Regarding temperature effect, results pointed to the importance of incubation temperature which regulated the biosynthesis of Cry4Ba and Cry411Aa complements differentially (Table 1). Cry4Ba toxin production was the highest at 25 ◦ C, a temperature generally considered to be low to permit a good growth in Bacillus spp. Growth and Cry11Aa synthesis were optimal at 37 ◦ C and 30 ◦ C, respectively. When studying the pH effect, 100 mM MOPS was included in the media to minimize pH changes. Cry4Ba formation was more susceptible to pH changes and interfered by alkaline pH. Sporulation frequency was highest when the cells were grown in pH 7.0 medium and the frequencies obtained at other pH values were about tenfold lower than this. It is to be noted that throughout this study Cyt1A yields were not remarkably influenced by any of the alterations in medium composition or cultivation conditions.
4. Discussion In striking contrast with commercial and scientific interest focused on B. thuringiensis israelensis is the lack of ad-
equate information on various aspects of toxin production by the organism in the published literature. It was this focal point that led us to investigate the most crucial cultural parameters for a typical B. thuringiensis israelensis batch fermentation to achieve high toxin titers. Electron microscopic studies have differentiated three different crystalline inclusions in B. thuringiensis israelensis [11]. Protein analyses have indicated that one of these crystals is made up of Cry11Aa polypeptide exclusively, and it has been suggested that the other toxin proteins should be assembled separately to form the other inclusions. In the present study, Cry4A and Cry4C proteins could not be shown in any of the israelensis strains (B. thuringiensis israelensis HD14, HD33 and 4Q2-72) possibly due to the expression of these proteins at subdetectable levels (data not shown). Indeed, Cry4Ba, Cry11Aa and Cyt1A proteins have always constituted predominant polypeptide bands in existing literature; Cry4A protein is occasionally seen and Cry4C is almost always absent, as remarked by Lereclus et al. [12]. It was interesting to find that (NH4 )2 HPO4 greatly stimulated Cry4Ba synthesis. Since such a stimulation could not be seen with other salts of ammonium, we perceived that the inorganic phosphate was the actual positive effector. Among other nitrogen sources tested, organic and inorganic, none had a beneficial effect. The nitrates specifically suppressed
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the synthesis of Cry4Ba protein with no effect on the other toxin complement. Toxin levels were fairly good and almost identical when the organism was grown on a variety of carbon sources including dextrin, whey, maltose, lactose, sucrose, inulin and glycerol. Poor production in glucose medium might suggest a “glucose effect” which is frequently encountered in secondary metabolite systems. On the other hand, the negative effect of starch and molasses is puzzling, since these carbon sources are not readily utilizable. The effect of glucose on Cry4A production was studied by Bhatnagar [2] at the mRNA level and it was found that the repressive effect of glucose was dependent on a phosphorylation step since protein kinase inhibitor calphostin C relieved protoxin synthesis at both the mRNA and protein level. When supplied in concentrations ranging from 0.3 to 300 mM, inorganic phosphate supports good growth; however, at concentrations higher than 10 mM, it greatly suppresses secondary metabolism [7]. It was therefore surprising that a high inorganic phosphate concentration was an absolute requirement for synthesis at a high level of antidipteran toxins, especially Cry4Ba protein. These observations were consistent with our earlier perception that the stimulatory effect of (NH4 )2 HPO4 is due to the phosphate component rather than the ammonium. In view of our results, this mineral should be provided at a 50 to 100 mM concentration for a feasible process. These optimum concentrations are 10- to 20-fold higher than that reported by Sikdar et al. [18] for the same subspecies; nonetheless the investigators have kept the concentration range narrower. In the study of Bhatnagar [3], the addition of exogenous inorganic phosphate (Pi) during resuspension was found to stimulate 135kD protoxin protein synthesis by B. thuringiensis israelensis HD522. Sikdar et al. [18] have recommended the inclusion of Fe and Cu which we found to be unnecessary in this study. The optimum concentration of Ca was reported as 10−2 M by the authors, a concentration that was at least two log units higher than our optimum. For Mg and Mn, their optimum values were comparable to those reported in this study. Striking alterations in secondary metabolite formation and/or sporulation by trace metal concentrations that do not affect vegetative growth have been experienced by many investigators. The yields of bacterial toxins are known to be greatly influenced by trace metals and other minerals, and in most cases it is not known whether the effect is a direct one or simply a manifestation of sporulation. As deduced from the results of the present work, it would be misleading to consider toxin formation solely as an event accompanying sporulation since there were examples of low levels of toxin production obtained from the cultures with very high levels of sporulation. In this respect, our findings supported the hypothesis of Rosa and Mignone [17] and Paramatha [15] that a high spore count is not enough to ensure a good toxicity. Heavy metal ranges selected in the present study were adapted from the previous documents to avoid insufficient or toxic concentrations [19,21]. Mn appeared
to be the most critical metal since a great increase was detected in yields of Cry4Ba and Cry11Aa toxins when Mn was supplied at 10−6 M, furthermore this element was not beneficial when present at lower or higher concentrations. In view of the results obtained in this study, surface response methodology will be used as an optimization approach [13] for five fermentation variables (dextrin concentration, Mn concentration, inorganic phosphate concentration, pH and temperature) each at five levels to examine them more thoroughly and determine the best conditions for both spore and toxin production.
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