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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 1168-1177© TÜBİTAKdoi:10.3906/biy-1512-38

High levels of polyphosphate kinase affect not only endotoxin production but also acid tolerance of Bacillus thuringiensis

Tuğrul DORUK1, Zeynep GİRGİN ERSOY1, Mehmet Salim ÖNCEL2, Sedef TUNCA GEDİK1,*1Department of Molecular Biology and Genetics, Faculty of Science, Gebze Technical University, Kocaeli, Turkey2Department of Environmental Engineering, Faculty of Engineering, Gebze Technical University, Kocaeli, Turkey

* Correspondence: sgedik@gtu.edu.tr

1. Introduction Inorganic phosphate is stored as a polyphosphate (polyP) polymer inside the cell. PolyP, which is found in every living cell, consists of hundreds of phosphate molecules joined together by high-energy anhydride bonds (Brown and Kornberg, 2004). Polyphosphate kinase (PPK; E.C. 2.7.4.1) is the enzyme responsible for the synthesis of polyP by using the terminal phosphate of ATP as a substrate (Ahn and Kornberg, 1990).

PolyP is known to play a very important role in different cellular processes in every living cell. For example, this polymer has been shown to play a crucial role in the regulation of adaptive responses of cells to physical and chemical stresses, including weak acid stress (Shiba et al., 2000; Price-Carter et al., 2005). It is also an important energy store used as ATP substitutes, and it is additionally used to regenerate NTP in association with adenylate kinases (Phillips et al., 1999). PolyP has been shown to be necessary for motility, biofilm formation, and other virulence properties of pathogenic bacteria (Ogawa et al., 2000; Rashid et al., 2000). The effect of polyP on the control of secondary metabolite biosynthesis by Streptomyces has also been

researched (Chouayekh and Virolle, 2002; Yalım Camcı et al., 2012).

Bacillus thuringiensis is the main agent used for biological control of insects in commercial agriculture and forest management. Insecticidal activity of B. thuringiensis is mainly due to the presence of several δ-endotoxins (crystal proteins), which are produced during the sporulation process. Medium ingredients and cultivation strategies, such as carbon and nitrogen sources, mineral elements, pH, and temperature, play important roles in the synthesis of these proteins (Sikdar et al., 1991; Morris et al., 1996; Icgen et al., 2002a, 2002b; El-Bendary, 2006; Okay and Özcengiz, 2011). We recently showed that polyP affects bioinsecticide biosynthesis by Bti. We obtained a recombinant strain (Bti ppk), which overproduced the ppk gene, and reported that the recombinant strain was about 8 times more toxic than the wild type against late second-instar laboratory-reared Culex quinquefasciatus larvae (Doruk et al., 2013). In the present study, this hypertoxic strain (Bti ppk) was characterized by its acid tolerance, phosphate uptake, and endotoxin production under different culture conditions.

Abstract: Inorganic phosphate is stored as a polyphosphate (polyP) polymer inside every living cell. This polymer is synthesized by the polyP kinase (PPK) enzyme using the terminal phosphate of ATP as substrate and it performs important functions in the cell. In this study, effects of high levels of PPK on Bacillus thuringiensis subsp. israelensis were analyzed. Recombinant Bti ppk, a PPK overproducer, was found to uptake more phosphate into the cell and produce a consistently higher amount of endotoxin than the wild type under culture conditions including a range of temperatures (25 °C, 30 °C, and 37 °C), pH values (pH 5, 6, 8, and 9), and carbon sources (maltose, mannitol, sucrose, and starch). Moreover, this strain was found to overexpress sigB, which might cause a significant increase in the acid tolerance of this microorganism. Spores of Bti ppk were found to be smaller compared to wild-type spores; however, bioassay experiments with third-instar wild Culex pipiens larvae proved that high toxicity is not the result of small spore size. This hypertoxic recombinant Bti strain is very useful for industrial applications, not only because it produces more endotoxin than the wild type under different culture conditions, but also because it is more acid-tolerant under the conditions tested. Key words: Polyphosphate kinase, ppk, Bacillus thuringiensis, endotoxin, bioinsecticide

Received: 16.12.2015 Accepted/Published Online: 26.02.2016 Final Version: 16.12.2016

Research Article

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2. Materials and methods2.1. Bacterial strainsBacillus thuringiensis subsp. israelensis (Bti) ATCC 35646 was kindly provided by Gwo-Chyuan Shaw (National Yang-Ming University, Taiwan). Bti pHT315, a control strain carrying an empty vector, and Bti ppk, a PPK overproducer strain, were constructed in our previous study (Doruk et al., 2013).2.2. Media and growth conditionsBti strains were grown in 100 mL of DSM (4 g/L nutrient broth (Merck), 25 mM K2HPO4, 25 mM KH2PO4, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 10 µM FeSO4, 10 µM MnCl2, and 5 g/L glucose) in a 500-mL flask at 30 °C (Donovan et al., 1988). Experiments were started from overnight cultures, which were diluted using spectrophotometric (OD600) measurements to equalize inoculum size. Erythromycin (25 µg/mL) was added to media to grow recombinant strains. Liquid cultures were aerated on a rotary shaker at 220 rpm.2.3. Protein isolationProteins were extracted according to the procedure of Donovan et al. (1988). Cells (10 mL, spore/crystal mixture) from cultures grown for 72 h were harvested and washed once with 1 M NaCl and twice with deionized water. The cells were then suspended in deionized water at 100 mg wet cell weight/mL. Fifty microliters of the cell suspension was added to 50 µL of lysis buffer [TE buffer (100 mM Tris-HCl, 20 mM EDTA, pH 7.4) containing 10 mg/mL lysozyme] and incubated at 37 °C for 30 min. After incubation, 10 µL of 2% sodium dodecyl sulfate (SDS) was added, and the suspension was vortexed for 30 s and centrifuged for 5 min in a microfuge. The pellet (spore/crystal mixture) was suspended in 50 µL of 0.2% SDS. Protein concentrations were determined with the BCA protein assay method with bovine serum albumin as standard (Smith et al., 1985). Equal amounts of spore/crystal mixture (60 µg) and molecular mass standards from Invitrogen (Carlsbad, CA, USA) were loaded on the 12% SDS-polyacrylamide gels, which were then stained with Coomassie Brilliant Blue R-250. TotalLab Quant

software (TotalLab, Newcastle-upon-Tyne, UK) was used to quantitate protein bands in the gels. 2.4. Gene expression analysis by RT-PCRThe InnuPREP RNA Mini Kit (Analytic Jena, Jena, Germany) was used to isolate total RNA. The RNA samples were treated with deoxyribonuclease I (Fermentas, Hanover, MD, USA) to remove DNA contamination. The AccuScript High-Fidelity 1st Strand cDNA Synthesis Kit (Stratagene, Santa Clara, CA, USA) was used for cDNA synthesis according to the manufacturer’s protocol. Negative controls were carried out with the primers (Table 1) and Pfu DNA polymerase to confirm the absence of contaminating DNA in the RNA preparations. The cDNA products were run on 0.8% agarose gel and were visualized and photographed under UV light using a digital imaging system (Vilber Lourmat, Eberhardzell, Germany). Primer specificity was checked using the Sort sequence locator program (Bikandi et al., 2004). Each amplified product was corroborated by direct sequencing of the PCR product.2.5. Measurement of phosphate uptakePhosphate uptake was determined by measuring the residual Pi content in culture supernatants with inductively coupled plasma optical emission spectrometry (ICP-OES) (Optima 7000 DV, PerkinElmer, Waltham, MA, USA). Culture supernatants were prepared as described by Chung et al. (2005) with some modifications. Equal inoculum sizes from overnight cultures were transferred to 100 mL of LB medium and grown to the early stationary phase (OD600 @ 6). Fifty milliliters of culture was then centrifuged at 4500 × g for 10 min. The pellet was washed with distilled water and the cells were transferred to 150 mL of fresh LB medium. Ten milliliters of sample was taken at 6 and 12 h of cultivation. These samples were centrifuged at 4500 × g for 10 min and then the Pi content of the supernatants was analyzed by ICP-OES. 2.6. Acid stress assayResistance of bacterial strains to acidity was measured as described by Ahn et al. (2009) with minor modifications. Cultures were grown to the midexponential phase (OD600 @ 0.6). An aliquot culture of 1.5 mL was washed once with

Table 1. Oligonucleotides used for RT-PCR.

Name of primer Sequence of primer Size of amplified product (bp)

Bti ppk F 5′-AGAGCAGCGAGAATTTATAG-3′538

Bti ppk R 5′-TAAATCTAATGGCCCATCCA-3′Bti 16S F 5′-AGCGGTGGAGCATGTGGTTTA-3′

476Bti 16S R 5′-GTGGTGTGACGGGCGGTGTGT-3′Bti SigB F 5′-AGTAGGCATGTTAGGGCTCTTAG-3′

509Bti SigB R 5′-AACCGTTCCCCTGTATCTTTTTGA-3′

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0.1 M glycine (pH 7.0) and the pellet was resuspended in 0.1 M glycine (pH 2.8). The cells were then incubated at 30 °C, and samples taken at 30, 60, and 90 min were serially diluted and plated on LB agar. After overnight incubation at 30 °C, cell viabilities (CFU: colony-forming units) were compared with time zero values. 2.7. Mosquito larvicidal activity Mosquito larvicidal activity was determined according to the method of Promdonkoy et al. (2005). A spore/crystal mixture isolated from each bacterial strain grown in DSM medium (for 72 h) was tested. Serially diluted spore/crystal mixtures were added to 2 mL of water containing 10 third-instar wild Culex pipiens larvae in each well of 24-well plates (well diameter: 1.5 cm). LC50 values were determined using probit analysis (Finney and Stevens, 1948) at the end of 24 h by taking the average results of three independent experiments (Table 2). 2.8. Scanning electron microscope analysisOvernight cultures (10 mL) of all strains were collected by centrifugation. Pellets were washed three times with deionized water and resuspended in deionized water. Five microliters of each sample was spotted onto a slide and allowed to dry at room temperature for about 15 min. Samples were coated with a gold layer (5 nm thick) with a sputter coater (Model BAL-TEC SCD 005 Sputter Coater, Balzers, Liechtenstein) to impart electrical conductivity. Visualization of the cells on the slides was performed using a Carl Zeiss EVO 40 model scanning electron microscope (SEM) instrument (Dresden, Germany). The accelerating voltage was 10 kV for all experiments. Several SEM pictures were taken, and size distributions were obtained by measuring the length and diameter of 100 individual spores (Table 3) according to the method of Carrera et al. (2007). ImageJ software (Schneider et al., 2012) was used for the measurements.

2.9. Statistical analysisEach experiment was repeated three times. Statistical analysis of experiments was carried out by means of the test for homogeneity of proportions, and significance of the treatment differences was analyzed with a nonparametric statistical test, the post hoc comparisons test (Marascuilo and McSweeney, 1967), at P ≤ 0.05 using SPSS (SPSS Inc., Chicago, IL, USA).

3. Results3.1. Acid resistance and phosphate uptake properties of hypertoxic Bti strainA number of studies have shown that polyP protects cells against reduced pH (McGrath and Quinn, 2000; Mullan et al., 2002; Moriarty et al., 2006). Acid tolerance of the hypertoxic Bti ppk strain was determined in the present study. The results indicated that Bti ppk is more acid-tolerant than the control strains (Figure 1). The population of viable control cells (Bti and Bti pHT315) was reduced by

Table 2. Mosquito larvicidal activity of Bti, Bti pHT315, and Bti ppk strains on third-instar Culex pipiens larvae. LC50 values were determined using probit analysis.

Strain Mosquito larvicidal activity(24 h) (LC50 ng/mL)* (mean ± SE)

Bti (wild type) 62 ± 0.707 (a)Bti pHT315 55.75 ± 2.25 (b)Bti ppk 6.5 ± 0.289 (c)

*LC50 is the concentration that causes 50% mortality.Different lowercase letters indicate that the means are statistically different at P ≤ 0.05 according to the post hoc multiple comparison test.

Table 3. Spore sizes of Bti, Bti pHT315, and Bti ppk strains. Values were obtained with ImageJ software using the adjust threshold command to obtain an optimum black and white image without losing information for at least 100 spores.

Length (µm) Height (µm) Volume (µm3)* Aspect ratio†

Bacteria Mean Range Mean Range Mean Mean

Bti (wild type) 1.449 ± 0.184 (a) 1.118–2.105 1.089 ± 0.137 (a) 0.789–1.579 0.916 ± 0.285 (a) 1.350 ± 0.233 (a)Bti pHT315 1.330 ± 0.167 (b) 0.987–1.776 1.101 ± 0.126 (a) 0.789–1.382 0.861 ± 0.256 (a) 1.219 ± 0.182 (b)Bti ppk 1.306 ± 0.145 (b) 0.987–1.711 1.127 ± 0.095 (a) 0.921–1.382 0.881 ± 0.209 (a) 1.162 ± 0.126 (b)

*Assuming that the spores are an ellipsoid, the volumes were calculated with the formula V = πLH2/6, in which L represents the length and H the diameter.†Dividing the length by the diameter of each individual spore, aspect ratios were calculated by mean of 100 spores.Different lowercase letters indicate that the means are statistically different at P ≤ 0.05 according to the post hoc multiple comparison test.

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approximately 4 log10 after incubation in buffer at pH 2.8 for 90 min. On the other hand, Bti ppk cells were reduced by about 2 log10 in the same experiment (Figure 1).

We also checked the expression level of the sigB gene (homologous to the rpoS gene, which acts as a sigma factor responsible for the activation of stress-induced genes) in recombinant and wild-type strains. Our transcriptional analysis results showed that the level of sigB expression was higher in the ppk-overproducing strain, which is more acid-tolerant than the wild-type strain at 48 h (Figure 2).

Phosphate uptakes by Bti ppk, control strain Bti pHT315 (empty plasmid), and wild-type Bti were analyzed using

ICP-OES (Figure 3). Statistical analysis of the data indicated that phosphate accumulation by Bti ppk was higher than that of both control strains (Figure 3a), although all the strains grew equally well in LB medium (Figure 3b). 3.2. Bioassay and SEM dataAccording to the bioassay results, the toxicity of the recombinant mutant strain (LC50 6.5 ± 0.289 ng/mL) against third-instar wild Culex pipiens larvae was found to be nearly 10 times higher than that of the wild type (LC50 62 ± 0.707 ng/mL) (Table 2). The LC50 value of the control strain (Bti pHT315) for the same larvae was 55.75 ± 2.25 ng/mL (Table 2).

If the spores of the ppk-overexpressing strain are smaller, then the proportion of toxin would be greater in this strain, although each cell produces the same amount of toxin. To eliminate this possibility, the spore size of each strain was determined using a SEM. As seen in Figure 4, spores of ppk-overexpressing cells (volume @ 0.881 µm3, aspect ratio @ 1.162 µm) and spores of Bti pHT315 (volume @ 0.861 µm3, aspect ratio @ 1.219 µm) are rounder and smaller when compared to wild-type spores (volume @ 0.916 mm3, aspect ratio @ 1.350 mm). These results proved that high toxicity is not the result of small spore size, since the spores of Bti pHT315 are also smaller compared to wild-type spores.3.3. Endotoxin production by mutant and control strainsViable cell and spore counts showed that all three strains (Bti, Bti pHT315, and Bti ppk) grew similarly in DSM medium (pH 7) at 30 °C (Doruk et al., 2013). Endotoxin production of Bti ppk was compared to that of Bti and Bti

Figure 1. Resistance of bacterial strains to acidity. Vertical bars indicate standard deviation from the mean value. Different lowercase letters indicate that the means are statistically different at P ≤ 0.05 according to the post hoc multiple comparison test. All the results are the means of three different experiments.

Figure 2. Comparison of expression levels of ppk and sigB. Samples were taken at 48 h of cultivation and mRNA expression levels were assessed by standard RT-PCR. 16S rRNA served as a loading control.

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pHT315 grown in DSM medium under a range of culture conditions. Endotoxin production of the ppk recombinant strain was higher compared to both Bti and Bti pHT315 at 25 °C, 30 °C, and 37 °C (Figure 5). Although the strains grew equally well at 42 °C, none of them produced endotoxin at this temperature (data not shown).

Protein profiles by SDS-PAGE of cells grown at pH 5, 6, 8, and 9 (initial pH values of the medium) in DSM medium at 30 °C showed that crystal protein production of Bti ppk was higher than that of both Bti and Bti pHT315 at each pH value studied (Figure 6).

When the cultures were grown at 30 °C in DSM (pH 7) containing maltose, mannitol, sucrose, or starch (each at a final concentration of 5 g/L) instead of glucose, endotoxin production levels were found to be significantly higher in the recombinant Bti strain than those in the control strains (Figure 7).

4. DiscussionBt, which is the best-known biological control agent of insects, was engineered in our previous study to have high levels of intracellular polyphosphate inside the cell by

Figure 3. (A) Phosphate uptake properties and (B) growth of Bti (triangle bars; ▲), Bti pHT315 (square bars; ), and Bti ppk (circle bars; ). Data are the average of three independent experiments. Different lowercase letters indicate that the means are statistically different at P ≤ 0.05 according to the post hoc multiple comparison test.

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overexpressing the ppk gene. An important increase (about 8 times) in the toxicity of Bti ppk against late second-instar laboratory-reared Culex quinquefasciatus was seen (Doruk et al., 2013).

A high intracellular concentration of polyP in the recombinant strain had a consistently stimulatory effect on endotoxin production when the cells were grown in DSM medium under a variety of temperature and pH conditions

Figure 4. Electron micrographs of the cells: (A) Bti (wild type), (B) Bti pHT315, and (C) Bti ppk.

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in this study. Furthermore, when maltose, mannitol, sucrose, and starch replaced glucose in the DSM medium, the recombinant Bti ppk strain continued to produce more endotoxin than the control strains. It is known that slowly utilized complex carbon sources often stimulate

secondary metabolite production (Drew and Demain, 1977). Although our starch experiment results support this information, regulatory mechanisms underlying the high-level synthesis of endotoxin need to be investigated. Bioassay results combined with SEM data proved that the

Figure 5. Effects of temperature on toxin production by Bti (wild type), Bti pHT315, and Bti ppk strains. The cells were grown on DSM medium with 75 mM Pi and at different temperatures: 25 °C (2, 3, 4), 30 °C (5, 6, 7), and 37 °C (8, 9, 10). Proteins were extracted after 72 h of incubation; lane 1, molecular weight standards (Fermentas); lanes 2, 5, and 8, Bti; lanes 3, 6, and 9, Bti pHT315; lanes 4, 7, and 10, Bti ppk. Sixty micrograms of total protein was loaded in each lane. The amount of protein was determined using TotalLab Quant (supplementary material, Table S1).

Figure 6. Effects of pH on toxin production of Bti (wild type), Bti pHT315, and Bti ppk strains. The cells were grown on DSM medium with 75 mM Pi and had different pH levels: pH 5 (2, 3, 4), pH 6 (5, 6, 7), pH 8 (8, 9, 10), and pH 9 (11, 12, 13). Proteins were extracted after 72 h of incubation; lane 1, molecular weight standards (Fermentas); lanes 2, 5, 8, and 11, Bti; lanes 3, 6, 9, and 12, Bti pHT315; lanes 4, 7, 10, and 13, Bti ppk. Sixty micrograms of total protein was loaded in each lane. The amount of protein was determined using TotalLab Quant (supplementary material, Table S2).

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Bti ppk strain produces more bioinsecticide than both the wild-type (Bti) and the vector-only (Bti pHT315) strains. Therefore, high toxicity is not the result of small spore size.

Environmental factors play a critical role in the toxin protein synthesis of B. thuringiensis. Some mineral elements, especially phosphate, have been shown to play a very important role in toxin production (Banerjee-Bhatnagar, 1999; Kurt et al., 2005). In all these studies, inorganic phosphate was added to culture media in different concentrations, and the stimulatory effect of phosphate on endotoxin production was evaluated. In the present study, phosphate uptake by the recombinant Bti ppk strain (overexpressing ppk) was found to be higher than that of both Bti and Bti pHT315, which could be one of the reasons for toxin overproduction by this strain.

During fermentation, pH fluctuations, and especially low pH values, cause cell injuries and death (Russell, 1992). Our data indicate that the high polyP content of this recombinant strain apparently increased the ability of the cells to survive under acidic conditions compared to the wild type. A number of studies have shown that polyP protects cells against reduced internal pH (McGrath and Quinn, 2000; Mullan et al., 2002; Moriarty et al., 2006). Although the precise physiological role of polyP at acidic pH levels is still unclear, Mullan et al. (2002) suggested that polyP provides a mechanism of pH homeostasis by acting as an intracellular cation trap and sequestering H+ ions. Since polyP is highly polyanionic, it is likely that this

polymer plays a buffering role inside the cell. The RNA polymerase subunit, RpoS (homologous to σB in gram-positive bacteria), acts as a sigma factor responsible for the activation of more than 50 stress-induced genes in E. coli (Loewen et al., 1998). It has been shown that σB-dependent general stress proteins have protective effects for the cells under several stress conditions (Engelmann and Hecker, 1996; Antelmann et al., 1997; Gaidenko and Price, 1998). PolyP affects the expression of rpoS in E. coli (Shiba et al., 1997) and weak acids are known to stimulate RpoS-mediated transcription (Price-Carter et al., 2005). Rao and Kornberg (1996) reported that the phenotypic features of the ppk mutant can be linked to decreased expression of the rpoS gene. Our transcriptional analysis results support these findings: increased levels of polyP protect Bti cells against reduced internal pH, most probably due to the increase of the expression of sigB in this microorganism.

Bti has been widely used in mosquito control programs. Although many efforts have been made by some groups to find low-cost media to produce bioinsecticides (Zouari et al., 1998; Ghribi et al., 2007), large-scale production of Bti in a cost-effective manner is still an attractive pursuit. The results of this study can be useful in industrial applications, since it could be possible to produce large quantities of insecticidal crystal proteins during large-scale fermentation, as the hypertoxic Bti ppk strain not only produces more endotoxin than the wild type under different conditions but also has the advantage of being more acid-tolerant.

Figure 7. Effects of different carbon sources on toxin production of Bti (wild type), Bti pHT315, and Bti ppk strains. The cells were grown on DSM medium with 75 mM Pi and had different carbon sources: sucrose (2, 3, 4), maltose (5, 6, 7), mannitol (8, 9, 10), and starch (11, 12, 13). Proteins were extracted after 72 h of incubation; lane 1, molecular weight standards (Fermentas); lanes 2, 5, 8, and 11, Bti; lanes 3, 6, 9, and 12, Bti pHT315; lanes 4, 7, 10, and 13, Bti ppk. Sixty micrograms of total protein was loaded in each lane. The amount of protein was determined using TotalLab Quant (supplementary material, Table S3).

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AcknowledgmentsThis work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK), Grant No. TBAG

(107T812). We would like to thank Dr Fikrettin Şahin and Yeditepe University for the SEM data. We are grateful to Dr Öner Koçak for his support with the bioassay experiments.

References

Ahn K, Kornberg A (1990). Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate. J Biol Chem 265: 11734-11739.

Ahn SJ, Ahn SJ, Browngardt CM, Burne RA (2009). Changes in biochemical and phenotypic properties of Streptococcus mutans during growth with aeration. Appl Environ Microb 75: 2517-2527.

Antelmann H, Engelmann S, Schmid R, Sorokin A, Lapidus A, Hecker M (1997). Expression of a stress- and starvation-induced dps/pexB-homologous gene is controlled by the alternative sigma factor sigmaB in Bacillus subtilis. J Bacteriol 179: 7251-7256.

Banerjee-Bhatnagar N (1999). Inorganic phosphate regulates CryIVA protoxin expression in Bacillus thuringiensis israelensis. Biochem Biophys Res Commun 262: 359-364.

Bikandi J, San Millan R, Fau-Rementeria A, Rementeria A, Fau-Garaizar J, Garaizar J (2004). In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction. Bioinformatics 20: 798-799.

Brown MR, Kornberg A (2004). Inorganic polyphosphate in the origin and survival of species. P Natl Acad Sci USA 101: 16085-16087.

Carrera M, Zandomeni RO, Fitzgibbon J, Sagripanti JL (2007). Difference between the spore sizes of Bacillus anthracis and other Bacillus species. J Appl Microbiol 102: 303-312.

Chouayekh H, Virolle MJ (2002). The polyphosphate kinase plays a negative role in the control of antibiotic production in Streptomyces lividans. Mol Microbiol 43: 919-930.

Chung H, Park M, Madhaiyan M, Seshadri S, Song J, Cho H, Sa T (2005). Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea. Soil Biol Biochem 37: 1970-1974.

Donovan WP, Gonzalez JM Jr, Gilbert MP, Dankocsik C (1988). Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene. Mol Genet Genomics 214: 365-372.

Doruk T, Avican U, Camci IY, Gedik ST (2013). Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis. Microbiol Res 168: 199-203.

Drew SW, Demain AL (1977). Effect of primary metabolites on secondary metabolism. Ann Rev Microbiol 31: 343-356.

El-Bendary MA (2006). Bacillus thuringiensis and Bacillus sphaericus biopesticides production. J Basic Microbiol 46: 158-170.

Engelmann S, Hecker M (1996). Impaired oxidative stress resistance of Bacillus subtilis sigB mutants and the role of katA and katE. FEMS Microbiol Lett 145: 63-69.

Finney DJ, Stevens WL (1948). A table for the calculation of working probits and weights in probit analysis. Biometrika 35: 191-201.

Gaidenko TA, Price CW (1998). General stress transcription factor sigmaB and sporulation transcription factor sigmaH each contribute to survival of Bacillus subtilis under extreme growth conditions. J Bacteriol 180: 3730-3733.

Ghribi D, Zouari N, Trigui W, Jaoua S (2007). Use of sea water as salts source in starch- and soya bean-based media, for the production of Bacillus thuringiensis bioinsecticides. Process Biochem 42: 374-378.

Icgen Y, Icgen B, Ozcengiz G (2002a). Regulation of crystal protein biosynthesis by Bacillus thuringiensis: I. Effects of mineral elements and pH. Res Microbiol 153: 599-604.

Icgen Y, Icgen B, Ozcengiz G (2002b). Regulation of crystal protein biosynthesis by Bacillus thuringiensis: II. Effects of carbon and nitrogen sources. Res Microbiol 153: 605-609.

Kurt A, Ozkan M, Ozcengiz G (2005). Inorganic phosphate has a crucial effect on Cry3Aa delta-endotoxin production. Lett Appl Microbiol 41: 303-308.

Loewen PC, Hu B, Strutinsky J, Sparling R (1998). Regulation in the rpoS regulon of Escherichia coli. Can J Microbiol 44: 707-717.

Marascuilo LA, McSweeney M (1967). Nonparametric post hoc comparisons for trend. Psychol Bull 67: 401-412.

McGrath JW, Quinn JP (2000). Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH. Appl Environ Microb 66: 4068-4073.

Moriarty TF, Mullan A, McGrath JW, Quinn JP, Elborn JS, Tunney MM (2006). Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates. Lett Appl Microbiol 42: 617-623.

Morris ON, Converse V, Kanagaratnam P, Davies JS (1996). Effect of cultural conditions on spore–crystal yield and toxicity of Bacillus thuringiensis subsp. aizawai (HD133). J Invertebr Pathol 67: 129-136.

Mullan A, Quinn JP, McGrath JW (2002). Enhanced phosphate uptake and polyphosphate accumulation in Burkholderia cepacia grown under low pH conditions. Microb Ecol 44: 69-77.

Ogawa N, Tzeng CM, Fraley CD, Kornberg A (2000). Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features. J Bacteriol 182: 6687-6693.

Okay S, Özcengiz G (2011). Molecular cloning, characterization, and homologous expression of an endochitinase gene from Bacillus thuringiensis serovar morrisoni. Turk J Biol 35: 1-7.

Phillips NF, Hsieh PC, Kowalczyk TH (1999). Polyphosphate glucokinase. Prog Mol Subcell Biol 23: 101-125.

DORUK et al. / Turk J Biol

1177

Price-Carter M, Fazzio TG, Vallbona EI, Roth JR (2005). Polyphosphate kinase protects Salmonella enterica from weak organic acid stress. J Bacteriol 187: 3088-3099.

Promdonkoy B, Promdonkoy P, Panyim S (2005). Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae. FEMS Microbiol Lett 252: 121-126.

Rao NN, Kornberg A (1996). Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli. J Bacteriol 178: 1394-1400.

Rashid MH, Rao NN, Kornberg A (2000). Inorganic polyphosphate is required for motility of bacterial pathogens. J Bacteriol 182: 225-227.

Russell JB (1992). Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling. J Appl Bacteriol 73: 363-370.

Schneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671-675.

Shiba T, Tsutsumi K, Ishige K, Noguchi T (2000). Inorganic polyphosphate and polyphosphate kinase: their novel biological functions and applications. Biochemistry (Mosc) 65: 315-323.

Shiba T, Tsutsumi K, Yano H, Ihara Y, Kameda A, Tanaka K, Takahashi H, Munekata M, Rao NN, Kornberg A (1997). Inorganic polyphosphate and the induction of rpoS expression. P Natl Acad Sci USA 94: 11210-11215.

Sikdar DP, Majumdar MK, Majumdar SK (1991). Effect of minerals on the production of the delta endotoxin by Bacillus thuringiensis subsp. israelensis. Biotechnol Lett 13: 511-514.

Smith P, Krohn RI, Hermanson G, Mallia A, Gartner F, Provenzano M, Fujimoto E, Goeke N, Olson B, Klenk D (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85.

Yalım Camcı İY, Doruk T, Avican Ü, Tunca Gedik S (2012). Deletion of polyphosphate kinase gene (ppk) has a stimulatory effect on actinorhodin production by Streptomyces coelicolor A3(2). Turk J Biol 36: 373-380.

Zouari N, Dhouib A, Ellouz R, Jaoua S (1998). Nutritional requirements of a strain of Bacillus thuringiensis subsp. kurstaki and use of gruel hydro for the formulation of a new medium for delta-endotoxin production. Appl Biochem Biotech 69: 41-52.

DORUK et al. / Turk J Biol

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Supplementary materials

Table S1. Effects of temperature on toxin production by Bti (wild type), Bti pHT315, and Bti ppk strains. Amounts of protein were determined using the TotalLab Quant program.

Cry4a (µg) Cry11a (µg) Cyt1a and/or Cyt2ab (µg)

Bti Bti pHT315 Bti ppk Bti Bti pHT315 Bti ppk Bti Bti pHT315 Bti ppk

25 °C 0.34 0.31 1.24 1.79 1.33 2.29 1.99 1.15 2.6730 °C 0.17 0.16 0.40 0.93 0.87 1.93 1.48 1.17 3.0537 °C 0.14 0.21 0.76 0.87 1.64 2.34 0.90 2.65 3.36

Table S2. Effects of pH on toxin production by Bti (wild type), Bti pHT315, and Bti ppk strains. Amounts of protein were determined using the TotalLab Quant program.

Cry4a (µg) Cry11a (µg) Cyt1a and/or Cyt2ab (µg)

Bti Bti pHT315 Bti ppk Bti Bti pHT315 Bti ppk Bti Bti pHT315 Bti ppk

pH 5 0.29 0.42 0.52 1.46 1.82 2.07 2.09 2.95 3.29pH 6 0.15 0.23 0.48 1.03 1.11 1.61 1.52 2.12 3.44pH 8 0.25 0.27 0.39 1.66 0.98 2.61 1.81 1.38 3.00pH 9 0.41 0.41 1.17 1.68 1.66 3.03 1.56 1.84 3.93

Table S3. Effects of different carbon sources on toxin production by Bti (wild type), Bti pHT315, and Bti ppk strains. Amounts of protein were determined using the TotalLab Quant program.

Cry4a (µg) Cry11a (µg) Cyt1a and/or Cyt2ab (µg)

Bti Bti pHT315 Bti ppk Bti Bti pHT315 Bti ppk Bti Bti pHT315 Bti ppk

Sucrose 0.09 0.26 0.32 0.53 1.20 1.51 1.50 2.52 3.72Mannitol 0.23 0.33 0.69 1.07 1.39 2.03 2.39 3.81 3.32Maltose 0.20 0.29 0.53 0.67 1.14 1.11 1.23 2.54 3.69Starch 0.29 0.32 0.81 1.49 1.63 3.30 3.19 3.73 6.21