Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6467-7
APPLIED MICROBIAL AND CELL PHYSIOLOGY
The distribution pattern of DNA and protoxin in Bacillus thuringiensis as revealed by laser confocal microscopy analysis Quanfang Hu & Jingfang Wang & Zujiao Fu & Xiangtao Mo & Xuezhi Ding & Liqiu Xia & Youming Zhang & Yunjun Sun
Received: 16 November 2014 / Revised: 6 February 2015 / Accepted: 8 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract It was reported that the parasporal crystal from Bacillus thuringiensis contained DNA fragments. To investigate the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages, a cry1Ac-gfp fusion gene was constructed and expressed in an acrystalliferous B. thuringiensis strain, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively. When the recombinant cells were at the vegetative growth stage, the Cry1Ac-GFP fusion protein was not expressed and the DNA fluorescent signal was evenly distributed throughout the cell. At the initial stage of sporulation, the Cry1Ac-GFP fusion protein was expressed and accumulated as inclusion body, while two condensed DNA signals existed at each pole of the cell. With the extension of culture time, it seemed that the DNA fluorescence from the region of spore development gradually became faint or vanishing, while the DNA signal was still present in the other pole or the remaining area of the mother cell. Interestingly and unexpectedly, there was no DNA fluorescence signal in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell, which might indicate that the DNA embodied in the Quanfang Hu and Jingfang Wang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6467-7) contains supplementary material, which is available to authorized users. Q. Hu : J. Wang : Z. Fu : X. Mo : X. Ding : L. Xia : Y. Sun (*) College of Life Science, Hunan Normal University, Changsha 410081, People’s Republic of China e-mail: [email protected] Z. Fu Hunan Institute of Microbiology, Changsha 410009, People’s Republic of China Y. Zhang State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China
inclusion body was not accessible to the DNA-specific dye. This was the first investigation devoted exclusively to the in vivo distribution of protoxin and DNA in B. thuringiensis at different growth stages. These data shed light on deeply understanding the process of sporulation and parasporal crystal formation as well as further exploring the interaction of DNA and protoxin in B. thuringiensis. Keywords Bacillus thuringiensis . Protoxin . DNA . In vivo distribution . Laser confocal microscopy
Introduction Bacillus thuringiensis is a Gram-positive soil bacterium that could produce various insecticidal protoxins with different activities and specificities (van Frankenhuyzen 2009). Generally, most B. thuringiensis protoxins were synthesized during the stationary phase and accumulated in the mother cell as parasporal crystals which could account for up to 25 % of the dry weight of the sporulated cells. It was estimated that each cell has to synthesize 106 to 2×106 protoxin molecules to form a crystal. A variety of mechanisms occurring at the transcriptional, posttranscriptional, and posttranslational levels were thought to contribute to such massive production of protoxins and their orderly deposition into a crystal (Agaisse and Lereclus 1995; Schnepf et al. 1998). It was reported that the parasporal crystals from B. thuringiensis subsp. kurstaki HD73 and other strains contained 20-kb heterologous double-stranded DNA fragments, which were composed of many different pieces of DNA with a size of 20 kb from the bacterial genome (Bietlot et al. 1993; Sun et al. 2007). These DNA fragments were an integral component of the parasporal crystal and they existed
Appl Microbiol Biotechnol
as 20-kb DNA-protoxin complex (Bietlot et al. 1993; Clairmont et al. 1998; Guo et al. 2011; Wu et al. 2012; Xia et al. 2005). A virus-like structure model was proposed for the DNA-protoxin complex based on the experimental data, in which the ellipsoid Cry1 protoxin molecules interact with the 20-kb DNA through the N-terminal toxic moiety while the C-terminal half of the molecule extends away from the central DNA core. The DNA seems to have an important role in the activation of protoxins as no activated toxin can be generated when the 20-kb DNA is removed from the DNAprotoxin complex (Clairmont et al. 1998; Schernthaner et al. 2002). It was speculated that the association of the protoxin with the DNA might be involved in crystal formation and probably facilitated the sequestering of the protoxin during sporulation (Clairmont et al. 1998). At present, though some reports indicated the presence of different 20-kb DNA fragments in the crystal and their roles in activation of protoxins, there were very few data comprehensively documenting the in vivo distribution pattern of protoxin and DNA in B. thuringiensis cells at different growth stages. A previous study using phase-contrast and fluorescence microscopy indicated that B. thuringiensis subsp. kurstaki HD-1 incubated with ethidium bromide showed a shifting pattern of nucleic acid distribution within the bacterium. Just before sporulation, nucleic acid condensed in the region where the spore formed. After spore formation, the fluorescence from this region vanished and appeared in the region where the crystalline inclusion body was assembled (Bietlot et al. 1993). Considering that laser confocal microscopy coupled to fluorescent reporter gene assay has been recently utilized to monitor protein localization and cell development (Ellermeier et al. 2006), we further investigated the distribution of protoxin and genomic DNA in B. thuringiensis at different growth stages in this study. A cry1Ac-gfp fusion gene was constructed under the control of the cry1Ac promoter and terminator and expressed in an acrystalliferous B. thuringiensis strain XBU001. The recombinant XBU001(pH1Ac-GFP) was collected at different culture time for observation with a laser confocal microscope, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively.
Materials and methods Bacterial strains and plasmids Escherichia coli DH5α and acrystalliferous B. thuringiensis strain XBU001 (CCTCC no. M2014452) (Yin et al. 2011) were used as host strains for transformation. B. thuringiensis subsp. kurstaki HD73 (obtained from Bacillus Genetic Stock Center, USA) was used for amplifying cry1Ac gene. E. coli GB05-Red was used for Red/ET homologous recombination
(Bian et al. 2012; Fu et al. 2012) while constructing a cry1Acgfp fusion gene. The plasmids pJP5603 (Penfold and Pemberton 1992) and pEGFP-N1 (Clontech) were the source of kanamycin resistance gene (kan) and GFP gene (gfp), respectively, and the E. coli-B. thuringiensis shuttle vector pHT315 (Arantes and Lereclus 1991) was used as a cloning vector. Construction of a cry1Ac-gfp fusion gene The cry1Ac-gfp fusion gene was constructed by fusing gfp to the 3′ terminus of cry1Ac. Primers 1Ac-F and 1Ac-R (Supplementary Table S1) were designed based on the published sequence (GenBank accession no. M11068) for amplifying the cry1Ac gene from B. thuringiensis strain HD73 (Adang et al. 1985). The 4.2-kb PCR fragment containing the promoter, the ORF, and the terminator of the cry1Ac gene was cloned into pHT315 between SalI and BamHI, producing recombinant plasmid pH1Ac. The kanamycin-resistant gene fragment was obtained from the plasmid pJP5603 by PCR using the primers Kan-F and Kan-R. The 5′ end of the 1.2-kb PCR product of kan gene has NcoI site and a 50-bp homology arm to the upstream sequence of the TAG stop codon of the cry1Ac gene, while its 3′ end has BglII site and a 50-bp homology arm to the downstream sequence of the TAG stop codon. Then, the circular plasmid pH1Ac and the linear PCR product of kan gene were transformed together into E. coli GB05-Red. By adding the arabinose to induce the occurrence of homologous recombination (Bian et al. 2012; Fu et al. 2012; Zhang et al. 2000), the TAG stop codon of the cry1Ac gene on the pH1Ac was substituted by the kan gene fragment flanked by NcoI and BglII, producing the kanamycin-resistant transformants harboring recombinant plasmid pH1AC-kan. By using this recombineering method, the NcoI and BglII sites were introduced between the ORF and the terminator of the cry1Ac gene. Subsequently, the primers GFP-F (containing the coding sequence of a flexible amino acid linker) and GFP-R were used to amplify the gfp gene from plasmid pEGFP-N1. The 0.7-kb PCR product was digested and then cloned into pH1AC-kan between NcoI and BglII to produce the recombinant plasmid pH1Ac-GFP. This plasmid contained the promoter and ORF of the cry1Ac gene without the stop codon, a flexible amino acid linker (GGGGSGGGGSGGGGS) coding sequence, gfp with its stop codon, and terminator of the cry1Ac gene. An outline describing the generation of the fusion gene and recombinant plasmids is shown in Supplementary Fig. S1. The plasmids pH1Ac-GFP and pH1Ac were transformed into the acrystalliferous B. thuringiensis strain XBU001 by electroporation (Park et al. 1998) to produce the recombinant strains XBU001(pH1Ac-GFP) and XBU001(pH1Ac), respectively. In our study, the most commonly used flexible amino acid linker (GGGGS)3 was
Appl Microbiol Biotechnol
adopted between Cry1Ac and GFP in order to reduce the negative effect of fusion on the conformation of Cry1Ac and GFP (Chen et al. 2013). Protein analysis B. thuringiensis recombinant strains were grown in G-Tris medium (Park et al. 1998) containing 25 μg/ml of erythromycin at 30 °C with shaking until cells lysed. The crystal-spore mixture was collected and resuspended in 0.5 M NaCl, sonicated for 5 min on ice, and then washed three times with deionized water. Then, the crystal-spore mixture was subjected to SDS-10 % PAGE analysis. In order to confirm the expressed protein, the predicted protein band on SDS-PAGE gel was sliced and digested by trypsin, and then subjected to LC-MS/MS analysis as described previously (Sun et al. 2008). Detection of DNA and fusion protein in the parasporal inclusions Parasporal inclusions from recombinant strain XBU001(pH1Ac-GFP) were purified as previously described by Pendleton and Morrison (1966), and then stored in aliquots at 4 °C. The purified inclusions were treated with DNase I (TaKaRa) for 15 min at 37 °C to degrade any DNA nonspecifically adsorbing to the inclusions. Subsequently, the DNase I-pretreated inclusions were solubilized in 50 mM bicarbonate/carbonate buffer (pH 10.5) containing 2 % (v/v) β-mercaptoethanol for 1 h at 4 °C. The suspension was centrifuged, and the supernatant was collected for analysis with 0.8 % (w/v) agarose gel electrophoresis and SDS-10 % PAGE, respectively.
Results Expression of Cry1Ac-GFP fusion protein The transcriptional fusion of cry1Ac-gfp cloned in the multicopy plasmid pHT315 was effectively expressed in the acrystalliferous B. thuringiensis host. SDS-PAGE analysis showed that XBU001(pH1Ac-GFP) and XBU001(pH1Ac) expressed a target protein band with the expected size of 160 and 130 kDa, respectively (Fig. 1). The 160-kDa target protein band from XBU001(pH1Ac-GFP) was excised from the gel and subjected to trypsin digestion. LC-MS/MS analysis of the tryptic fragments and subsequent database searching confirmed that this protein band was the fusion product of Cry1Ac and GFP (Supplementary Table S2). Observation with laser confocal microscope showed that the Cry1AcGFP fusion protein could form large parasporal inclusion body in B. thuringiensis host (Fig. 2). Meanwhile, we found that there existed some protein bands less than 160 kDa for XBU001(pH1Ac-GFP), which were derived from degraded products of the fusion protein as indicated by Western blot analysis using Cry1Ac antibody and GFP antibody (data not shown). Such result might suggest that the fusion protein possessed lower stability and was liable to be degraded while preparing SDS-PAGE sample.
Laser confocal microscopy The synchronously dividing B. thuringiensis cells prepared as described by Bechtel and Bulla (1976) were inoculated into G-Tris medium and cultured at 30 °C with shaking. B. thuringiensis cells cultured for different period were collected and stained with DNA-specific dye Hoechst 33258 (Invitrogen) according to the protocol provided by the manufacturer. Then, the stained cells were observed using laser confocal microscope (Zeiss LSM 710). The localization of DNA and protoxin in B. thuringiensis cells was monitored by observing the blue and green fluorescence signals from Hoechst 33258 a n d G F P. F o r s t a i n i n g w i t h e t h i d iu m b r o m i d e , B. thuringiensis cells were incubated for 30 min at room temperature with ethidium bromide at 1 μg/ml in Tris/acetic acid/EDTA, and then scanned using a laser confocal microscope.
Fig. 1 SDS-PAGE analysis of crystal-spore mixture from sporulated B. thuringiensis strains. Lane M, molecular mass marker; lane 1, recombinant strain XBU001(pH1Ac-GFP); lane 2, strain XBU001(pH1Ac). Cry1Ac-GFP fusion protein and Cry1Ac are indicated by arrows
Appl Microbiol Biotechnol Fig. 2 Distribution of Cry1AcGFP fusion protein and DNA in the recombinant strain XBU001(pH1Ac-GFP) at different growth stages in G-Tris medium. The cells cultured for 5.5, 10.5, 12.5, 14.5, 18.5, and 34 h, respectively, were stained with Hoechst 33258 and observed with a laser confocal microscope. GFP green fluorescent protein signal in the bacterial cells, Hoechst blue fluorescent signal of the Hoechst 33258 dye, DIC differential interference contrast microscopy. The overlay shows overlap of GFP, Hoechst, and DIC graphs. Yellow arrows point to DNA in the cells; red arrows point to inclusion body of Cry1Ac-GFP fusion protein; white arrows point to developing and mature spores. Bar, 2 μm
Solubilization of the parasporal inclusions When the parasporal inclusions from XBU001(pH1Ac-GFP) were pretreated with DNase I and then incubated in 50 mM bicarbonate/carbonate buffer (pH 10.5) containing 2 % (v/v) β-mercaptoethanol, the inclusions were solubilized and the released 160-kDa protein was detected on SDS-PAGE, which was confirmed to be Cry1Ac-GFP fusion protein by in-gel tryptic digestion and LC-MS/MS analysis (Supplementary
Table S2). A DNA band with molecular size of approximate 20 kb could be detected when the solubilized fusion protein was subjected to agarose gel electrophoresis (Supplementary Fig. S2). Distribution of Cry1Ac-GFP and DNA in B. thuringiensis In order to monitor the distribution of protoxin and DNA in B. thuringiensis cells, the recombinant strain
Appl Microbiol Biotechnol
XBU001(pH1Ac-GFP) was cultured in G-Tris medium and analyzed by laser confocal microscopy at different growth stages (Fig. 2). A green fluorescent signal indicated the production and accumulation of the Cry1Ac-GFP fusion protein, while a blue fluorescent signal from Hoechst 33258 indicated the distribution of DNA in the cell. When XBU001(pH1AcFig. 3 Distribution of wild Cry1Ac protoxin and DNA in the recombinant strain XBU001(pH1Ac) at different growth stages in G-Tris medium. The cells cultured for 5.5, 10.5, 14.5, 18.5, and 34 h, respectively, were stained with Hoechst 33258 and observed with a laser confocal microscope. Hoechst blue fluorescent signal of the Hoechst 33258 dye, DIC differential interference contrast microscopy. The overlay shows overlap of Hoechst and DIC graphs. Yellow arrows point to DNA in the cells; red arrows point to crystalline inclusion body of Cry1Ac protoxin; white arrows point to developing and mature spores. Bar, 2 μm
GFP) was grown for 5.5 h, the cells were at the vegetative growth stage. No green fluorescent signal was observed in B. thuringiensis cells, while the DNA was found to be evenly distributed in the cell (yellow arrows). When grown for 10.5 and 12.5 h, GFP signal started to appear in the recombinant cells and generally concentrated as single particle (red
Appl Microbiol Biotechnol
arrows), indicating that the fusion protein was expressed and accumulated as inclusion body. The green fluorescent signal intensity gradually increased with the extension of culture time. Meanwhile, there were two condensed DNA fluorescence signals located in the pole of the cells (yellow arrows). At this stage, no visible spore could be observed. When cultured for 14.5 and 18.5 h, the spore in the recombinant cells could be distinguished (white arrows). DNA signal from the region of spore formation gradually became faint or vanishing with the development of spore, while the other pole or remaining area around the spore and inclusion in the cells still had strong DNA signal. From the very beginning of Cry1Ac-GFP expression to the maturation of inclusion body, it seemed that the DNA fluorescent signal did not appear in the region where the Cry1Ac-GFP inclusion body was assembled. When cultured for 34 h, the spore and inclusion body were released from the lysed cells. The released inclusion body did not exhibit DNA fluorescent signal but part of spores showed very faint DNA signal. Observation of wild Cry1Ac production and DNA distribution in B. thuringiensis As we were concerned that the distribution pattern of Cry1Ac protoxin and DNA in B. thuringiensis cells might be altered owing to the fusion of Cry1Ac and GFP, we further investigated wild Cry1Ac production and DNA distribution in the recombinant strain XBU001(pH1Ac) cultured in G-Tris medium by using DNA-specific dye Hoechst 33258 and laser confocal microscopy (Fig. 3). It was found that there was no obvious difference in growth curve and sporulation efficiency between strains XBU001(pH1Ac-GFP) and XBU001(pH1Ac) (data not shown). When cultured for 5.5 h, the DNA was evenly distributed in the cells. At 10.5 h, there appeared two condensed DNA signals in each pole of the cells (yellow arrows). When cultured for 14.5 and 18.5 h, the spore appeared at one end of the cells (white
Fig. 4 Distribution of wild Cry1Ac protoxin and DNA in the mature unlysed XBU001(pH1Ac) cultured for 18.5 h in G-Tris medium. The cells were stained with ethidium bromide and observed with a laser confocal microscope. EB orange fluorescent signal of the ethidium
arrows) and the bipyramidal crystals of Cry1Ac could be gradually distinguished (red arrows). The spore emitted faint DNA fluorescence while the other end still existed strong DNA signal at 14.5 h. However, the DNA fluorescence in the spore region vanished at 18.5 h and there existed DNA signals in the remaining area of the cells. It was noteworthy that the bipyramidal crystals of Cry1Ac did not exhibit DNA fluorescence. When cultured for 34 h, the spore and crystal were released from the lysed cells and not fluorescent on Hoechst 33258 staining. Furthermore, we treated the sporulated XBU001(pH1Ac) cells with another nucleic acid dye ethidium bromide and visualized them by laser confocal microscopy. It was found that there was no DNA fluorescence from the bipyramidal crystal of Cry1Ac (Fig. 4), which was consistent with the results obtained by Hoechst 33258 staining. The distribution pattern of protoxin and DNA in strain XBU001(pH1Ac) at the sampling time seemed to be similar to that observed in XBU001(pH1Ac-GFP). However, the expression and distribution of Cry1Ac could not be clearly observed before the Cry1Ac protoxins were accumulated as visible inclusion. In contrast, even the small particle formed by Cry1Ac-GFP could be clearly distinguished by GFP signal at the very beginning of expression. For example, the Cry1AcGFP was expressed and accumulated as tiny particle when cultured for 10.5 h, while the expression of wild Cry1Ac could not be monitored in XBU001(pH1Ac) at this time.
Discussion One of the most striking features of B. thuirngiensis is the synthesis of the parasporal crystal during sporulation. The genomic DNA in B. thuringiensis not only direct the formation of spore but also the protoxin synthesis. Especially, it was reported that heterologous 20-kb DNA fragments were intimately associated with the parasporal crystal and they existed
bromide dye, DIC differential interference contrast microscopy. The overlay shows overlap of EB and DIC graphs. Yellow arrows point to DNA in the cells; red arrows point to crystalline inclusion body of Cry1Ac protoxin; white arrows point to spores. Bar, 2 μm
Appl Microbiol Biotechnol
as 20-kb DNA-protoxin complex (Bietlot et al. 1993; Schnepf et al. 1998). In this study, we explored the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages utilizing fluorescent reporter GFP and laser confocal microscopy. The shifting pattern of DNA distribution and the accumulation of Cry1Ac-GFP within the B. thuringiensis cell were clearly presented. Meanwhile, we did not observe co-localization of GFP signal from parasporal inclusion body with DNA fluorescence signal at different growth stages, which conflicted with the previous report that DNA fluorescence appeared in the region where the crystalline inclusion body was assembled in B. thuringiensis (Bietlot et al. 1993). The Cry1Ac-GFP fusion protein was constructed to indicate the cellular localization of Cry1Ac protoxin in this study. As indicated by laser confocal microscopic analysis, the Cry1Ac-GFP fusion protein was effectively expressed in B. thuringiensis host, which might be due to the fact that the transcriptional fusion of cry1Ac-gfp was cloned into the multicopy shuttle vector pHT315 (10 to 15 copies per cell) under the control of the overlapping promoters (BtI and BtII) and the transcription terminator with two stem-loop structures of the cry1Ac gene (Agaisse and Lereclus 1995). These genetic elements could promote transcription of the fusion gene and stability of the produced mRNA. In addition, the deposition of this fusion protein into parasporal inclusion body might decrease its susceptibility to premature proteolytic degradation in B. thuringiensis host. However, the Cry1Ac-GFP fusion protein could not form bipyramidal inclusion as native Cry1Ac, possibly because fusion to GFP interfered with the folding of fusion protein to some extent. Nevertheless, the inclusion body could be solubilized in the alkaline solution and the released fusion protein was associated with DNA. Extensive studies of the sporulation of Bacillus subtilis have provided detailed information on the phenotypes of such differentiation process (Ben-Yehuda et al. 2003; Errington 1993), which was helpful for explaining the distribution pattern of protoxin and DNA in B. thuringiensis. B. subtilis cells that have begun to sporulate contain two condensed chromosomes known as the axial filament, which extends from pole to pole with the origins located at its ends. A septum then forms near one pole and divides the developing cell unequally into a forespore and a mother cell, and then a DNA translocase would pump one chromosome across the septum into the forespore. Our study showed that there existed two strong DNA signals at the pole of the B. thuringiensis cells at the early stages of sporulation, which might correspond to two condensed genomic DNA. One DNA mass would be trapped in the forespore and another would stay in the mother cell. In addition, there existed some space between the two DNA fluorescent regions with nearly round shape. As indicated by our observation, such space seemed to be the region where Cry1Ac-GFP fusion protein was initially deposited as inclusion body. At the subsequent stages of spore formation, the
DNA fluorescence from the region of spore development became faint and ultimately vanished, possibly suggesting that the DNA was gradually enveloped by the developing spore and then totally covered by the mature spore. As to the DNA mass in the mother cell, it gradually became scattered and moved from the pole to the space around the spore and inclusion body with the extension of culture time. During this process, perhaps the produced Cry1Ac-GFP fusion proteins would somehow take up pieces of 20-kb DNA derived from genomic DNA. Interestingly and unexpectedly, we found that there was no DNA fluorescence in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell. Our study using the strain XBU001(pH1Ac) treated with Hoechst 33258 also showed that the DNA fluorescence did not appear in the region where the crystal of Cry1Ac was assembled. However, a previous report (Bietlot et al. 1993) observed a fluorescence signal of DNA from the region of mature crystal in B. thuringiensis subsp. kurstaki HD1 incubated with ethidium bromide under fluorescence microscope. Even though we treated XBU001(pH1Ac) with ethidium bromide, we also did not observe DNA fluorescence from the mature crystal of Cry1Ac using laser confocal microscope. The reason for the discrepancy between the previous report and our results was not known yet. We speculated that the DNA fluorescence previously observed in the region of mature crystal with fluorescence microscope might originate from free DNA surrounding the crystal. The DNA in the growing and mature inclusion body in B. thuringiensis cell could not be seen when staining with DNA-specific dye, possibly because the parasporal inclusion body possessed compact structure and completely covered the embodied DNA, thus preventing access of the fluorescent dye to the DNA. Acknowledgments This research was supported by grants from National Natural Science Foundation of China (30900037), State Key Laboratory of Microbial Technology (M2013-04), Hunan Provincial Natural Science Foundation (12JJ6024, 09JJ3078), and Program for Excellent Talents in Hunan Normal University (ET13105), the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), People’s Republic of China.
References Adang MJ, Staver MJ, Rocheleau TA, Leighton J, Barker RF, Thompson DV (1985) Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensissubsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene 36:289–300 Agaisse H, Lereclus D (1995) How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177:6027–6032 Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119. doi:10.1016/03781119(91)90495-W
Appl Microbiol Biotechnol Bechtel DB, Bulla LA Jr (1976) Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis. J Bacteriol 127:1472–1481 Ben-Yehuda S, Rudner DZ, Losick R (2003) RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299:532–536 Bian X, Huang F, Stewart FA, Xia L, Zhang Y, Müller R (2012) Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering. Chembiochem 13:1946–1952. doi:10.1002/cbic. 201200310 Bietlot HP, Schernthaner JP, Milne RE, Clairmont FR, Bhella RS, Kaplan H (1993) Evidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA. J Biol Chem 268:8240–8245 Chen X, Zaro JL, Shen WC (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65:1357–1369. doi: 10.1016/j.addr.2012.09.039 Clairmont FR, Milne RE, Pham VT, Carrière MB, Kaplan H (1998) Role of DNA in the activation of the Cry1A insecticidal crystal protein from Bacillus thuringiensis. J Biol Chem 273:9292–9296. doi:10. 1074/jbc.273.15.9292 Ellermeier CD, Hobbs EC, Gonzalez-Pastor JE, Losick R (2006) A threeprotein signaling pathway governing immunity to a bacterial cannibalism toxin. Cell 124:549–559. doi:10.1016/j.cell.2005.11.041 Errington J (1993) Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57:1–33 Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Müller R, Stewart AF, Zhang Y (2012) Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol 30:440–446. doi:10.1038/ nbt.2183 Guo S, Li J, Liu Y, Song F, Zhang J (2011) The role of DNA binding with the Cry8Ea1 toxin of Bacillus thuringiensis. FEMS Microbiol Lett 317:203–210. doi:10.1111/j.1574-6968.2011.02230.x Park HW, Ge B, Bauer LS, Federici BA (1998) Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64:3932–3938 Pendleton IR, Morrison RB (1966) Separation of the spores and crystals of Bacillus thuringiensis. Nature 212:728–729
Penfold RJ, Pemberton JM (1992) An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118: 145–146. doi:10.1016/0378-1119(92)90263-O Schernthaner JP, Milne RE, Kaplan H (2002) Characterization of a novel insect digestive DNase with a highly alkaline pH optimum. Insect Biochem Mol Biol 32:255–263. doi:10.1016/S0965-1748(01) 00084-4 Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806 Sun Y, Fu Z, Ding X, Xia L (2008) Evaluating the insecticidal genes and their expressed products in Bacillus thuringiensis strains by combining PCR with mass spectrometry. Appl Environ Microbiol 74:6811– 6813. doi:10.1128/AEM. 01085-08 Sun Y, Wei W, Ding X, Xia L, Yuan Z (2007) Detection of chromosomally located and plasmid-borne genes on 20 kb DNA fragments in parasporal crystals from Bacillus thuringiensis. Arch Microbiol 188: 327–332. doi:10.1007/s00203-007-0252-7 van Frankenhuyzen K (2009) Insecticidal activity of Bacillus thuringiensis crystal proteins. J Invertebr Pathol 101:1–16. doi:10. 1016/j.jip.2009.02.009 Wu F, Zhao X, Sun Y, Li W, Xia L, Ding X, Yin J, Hu S, Yu Z, Tang Y (2012) Construction of gene library of 20 kb DNAs from parasporal crystal in Bacillus thuringiensis strain 4.0718: phylogenetic analysis and molecular docking. Curr Microbiol 64:106–111. doi:10.1007/ s00284-011-0038-7 Xia L, Sun Y, Ding X, Fu Z, Mo X, Zhang H, Yuan Z (2005) Identification of cry-type genes on 20-kb DNA associated with Cry1 crystal proteins from Bacillus thuringiensis. Curr Microbiol 51:53–58. doi:10.1007/s00284-005-4504-y Yin J, Ding X, Xia L, Yu Z, Lv Y, Hu S, Huang S, Cao Z, Xiao X (2011) Transcription of gene in an acrystalliferous strain of Bacillus thuringiensis XBU001 positively regulated by the metalloprotease camelysin gene at the onset of stationary phase. FEMS Microbiol Lett 318:92–100. doi:10.1111/j.1574-6968.2011.02247.x Zhang Y, Muyrers JP, Testa G, Stewart AF (2000) DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol 18:1314–1317. doi:10.1038/82449
APPLIED MICROBIAL AND CELL PHYSIOLOGY
The distribution pattern of DNA and protoxin in Bacillus thuringiensis as revealed by laser confocal microscopy analysis Quanfang Hu & Jingfang Wang & Zujiao Fu & Xiangtao Mo & Xuezhi Ding & Liqiu Xia & Youming Zhang & Yunjun Sun
Received: 16 November 2014 / Revised: 6 February 2015 / Accepted: 8 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract It was reported that the parasporal crystal from Bacillus thuringiensis contained DNA fragments. To investigate the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages, a cry1Ac-gfp fusion gene was constructed and expressed in an acrystalliferous B. thuringiensis strain, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively. When the recombinant cells were at the vegetative growth stage, the Cry1Ac-GFP fusion protein was not expressed and the DNA fluorescent signal was evenly distributed throughout the cell. At the initial stage of sporulation, the Cry1Ac-GFP fusion protein was expressed and accumulated as inclusion body, while two condensed DNA signals existed at each pole of the cell. With the extension of culture time, it seemed that the DNA fluorescence from the region of spore development gradually became faint or vanishing, while the DNA signal was still present in the other pole or the remaining area of the mother cell. Interestingly and unexpectedly, there was no DNA fluorescence signal in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell, which might indicate that the DNA embodied in the Quanfang Hu and Jingfang Wang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6467-7) contains supplementary material, which is available to authorized users. Q. Hu : J. Wang : Z. Fu : X. Mo : X. Ding : L. Xia : Y. Sun (*) College of Life Science, Hunan Normal University, Changsha 410081, People’s Republic of China e-mail: [email protected] Z. Fu Hunan Institute of Microbiology, Changsha 410009, People’s Republic of China Y. Zhang State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China
inclusion body was not accessible to the DNA-specific dye. This was the first investigation devoted exclusively to the in vivo distribution of protoxin and DNA in B. thuringiensis at different growth stages. These data shed light on deeply understanding the process of sporulation and parasporal crystal formation as well as further exploring the interaction of DNA and protoxin in B. thuringiensis. Keywords Bacillus thuringiensis . Protoxin . DNA . In vivo distribution . Laser confocal microscopy
Introduction Bacillus thuringiensis is a Gram-positive soil bacterium that could produce various insecticidal protoxins with different activities and specificities (van Frankenhuyzen 2009). Generally, most B. thuringiensis protoxins were synthesized during the stationary phase and accumulated in the mother cell as parasporal crystals which could account for up to 25 % of the dry weight of the sporulated cells. It was estimated that each cell has to synthesize 106 to 2×106 protoxin molecules to form a crystal. A variety of mechanisms occurring at the transcriptional, posttranscriptional, and posttranslational levels were thought to contribute to such massive production of protoxins and their orderly deposition into a crystal (Agaisse and Lereclus 1995; Schnepf et al. 1998). It was reported that the parasporal crystals from B. thuringiensis subsp. kurstaki HD73 and other strains contained 20-kb heterologous double-stranded DNA fragments, which were composed of many different pieces of DNA with a size of 20 kb from the bacterial genome (Bietlot et al. 1993; Sun et al. 2007). These DNA fragments were an integral component of the parasporal crystal and they existed
Appl Microbiol Biotechnol
as 20-kb DNA-protoxin complex (Bietlot et al. 1993; Clairmont et al. 1998; Guo et al. 2011; Wu et al. 2012; Xia et al. 2005). A virus-like structure model was proposed for the DNA-protoxin complex based on the experimental data, in which the ellipsoid Cry1 protoxin molecules interact with the 20-kb DNA through the N-terminal toxic moiety while the C-terminal half of the molecule extends away from the central DNA core. The DNA seems to have an important role in the activation of protoxins as no activated toxin can be generated when the 20-kb DNA is removed from the DNAprotoxin complex (Clairmont et al. 1998; Schernthaner et al. 2002). It was speculated that the association of the protoxin with the DNA might be involved in crystal formation and probably facilitated the sequestering of the protoxin during sporulation (Clairmont et al. 1998). At present, though some reports indicated the presence of different 20-kb DNA fragments in the crystal and their roles in activation of protoxins, there were very few data comprehensively documenting the in vivo distribution pattern of protoxin and DNA in B. thuringiensis cells at different growth stages. A previous study using phase-contrast and fluorescence microscopy indicated that B. thuringiensis subsp. kurstaki HD-1 incubated with ethidium bromide showed a shifting pattern of nucleic acid distribution within the bacterium. Just before sporulation, nucleic acid condensed in the region where the spore formed. After spore formation, the fluorescence from this region vanished and appeared in the region where the crystalline inclusion body was assembled (Bietlot et al. 1993). Considering that laser confocal microscopy coupled to fluorescent reporter gene assay has been recently utilized to monitor protein localization and cell development (Ellermeier et al. 2006), we further investigated the distribution of protoxin and genomic DNA in B. thuringiensis at different growth stages in this study. A cry1Ac-gfp fusion gene was constructed under the control of the cry1Ac promoter and terminator and expressed in an acrystalliferous B. thuringiensis strain XBU001. The recombinant XBU001(pH1Ac-GFP) was collected at different culture time for observation with a laser confocal microscope, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively.
Materials and methods Bacterial strains and plasmids Escherichia coli DH5α and acrystalliferous B. thuringiensis strain XBU001 (CCTCC no. M2014452) (Yin et al. 2011) were used as host strains for transformation. B. thuringiensis subsp. kurstaki HD73 (obtained from Bacillus Genetic Stock Center, USA) was used for amplifying cry1Ac gene. E. coli GB05-Red was used for Red/ET homologous recombination
(Bian et al. 2012; Fu et al. 2012) while constructing a cry1Acgfp fusion gene. The plasmids pJP5603 (Penfold and Pemberton 1992) and pEGFP-N1 (Clontech) were the source of kanamycin resistance gene (kan) and GFP gene (gfp), respectively, and the E. coli-B. thuringiensis shuttle vector pHT315 (Arantes and Lereclus 1991) was used as a cloning vector. Construction of a cry1Ac-gfp fusion gene The cry1Ac-gfp fusion gene was constructed by fusing gfp to the 3′ terminus of cry1Ac. Primers 1Ac-F and 1Ac-R (Supplementary Table S1) were designed based on the published sequence (GenBank accession no. M11068) for amplifying the cry1Ac gene from B. thuringiensis strain HD73 (Adang et al. 1985). The 4.2-kb PCR fragment containing the promoter, the ORF, and the terminator of the cry1Ac gene was cloned into pHT315 between SalI and BamHI, producing recombinant plasmid pH1Ac. The kanamycin-resistant gene fragment was obtained from the plasmid pJP5603 by PCR using the primers Kan-F and Kan-R. The 5′ end of the 1.2-kb PCR product of kan gene has NcoI site and a 50-bp homology arm to the upstream sequence of the TAG stop codon of the cry1Ac gene, while its 3′ end has BglII site and a 50-bp homology arm to the downstream sequence of the TAG stop codon. Then, the circular plasmid pH1Ac and the linear PCR product of kan gene were transformed together into E. coli GB05-Red. By adding the arabinose to induce the occurrence of homologous recombination (Bian et al. 2012; Fu et al. 2012; Zhang et al. 2000), the TAG stop codon of the cry1Ac gene on the pH1Ac was substituted by the kan gene fragment flanked by NcoI and BglII, producing the kanamycin-resistant transformants harboring recombinant plasmid pH1AC-kan. By using this recombineering method, the NcoI and BglII sites were introduced between the ORF and the terminator of the cry1Ac gene. Subsequently, the primers GFP-F (containing the coding sequence of a flexible amino acid linker) and GFP-R were used to amplify the gfp gene from plasmid pEGFP-N1. The 0.7-kb PCR product was digested and then cloned into pH1AC-kan between NcoI and BglII to produce the recombinant plasmid pH1Ac-GFP. This plasmid contained the promoter and ORF of the cry1Ac gene without the stop codon, a flexible amino acid linker (GGGGSGGGGSGGGGS) coding sequence, gfp with its stop codon, and terminator of the cry1Ac gene. An outline describing the generation of the fusion gene and recombinant plasmids is shown in Supplementary Fig. S1. The plasmids pH1Ac-GFP and pH1Ac were transformed into the acrystalliferous B. thuringiensis strain XBU001 by electroporation (Park et al. 1998) to produce the recombinant strains XBU001(pH1Ac-GFP) and XBU001(pH1Ac), respectively. In our study, the most commonly used flexible amino acid linker (GGGGS)3 was
Appl Microbiol Biotechnol
adopted between Cry1Ac and GFP in order to reduce the negative effect of fusion on the conformation of Cry1Ac and GFP (Chen et al. 2013). Protein analysis B. thuringiensis recombinant strains were grown in G-Tris medium (Park et al. 1998) containing 25 μg/ml of erythromycin at 30 °C with shaking until cells lysed. The crystal-spore mixture was collected and resuspended in 0.5 M NaCl, sonicated for 5 min on ice, and then washed three times with deionized water. Then, the crystal-spore mixture was subjected to SDS-10 % PAGE analysis. In order to confirm the expressed protein, the predicted protein band on SDS-PAGE gel was sliced and digested by trypsin, and then subjected to LC-MS/MS analysis as described previously (Sun et al. 2008). Detection of DNA and fusion protein in the parasporal inclusions Parasporal inclusions from recombinant strain XBU001(pH1Ac-GFP) were purified as previously described by Pendleton and Morrison (1966), and then stored in aliquots at 4 °C. The purified inclusions were treated with DNase I (TaKaRa) for 15 min at 37 °C to degrade any DNA nonspecifically adsorbing to the inclusions. Subsequently, the DNase I-pretreated inclusions were solubilized in 50 mM bicarbonate/carbonate buffer (pH 10.5) containing 2 % (v/v) β-mercaptoethanol for 1 h at 4 °C. The suspension was centrifuged, and the supernatant was collected for analysis with 0.8 % (w/v) agarose gel electrophoresis and SDS-10 % PAGE, respectively.
Results Expression of Cry1Ac-GFP fusion protein The transcriptional fusion of cry1Ac-gfp cloned in the multicopy plasmid pHT315 was effectively expressed in the acrystalliferous B. thuringiensis host. SDS-PAGE analysis showed that XBU001(pH1Ac-GFP) and XBU001(pH1Ac) expressed a target protein band with the expected size of 160 and 130 kDa, respectively (Fig. 1). The 160-kDa target protein band from XBU001(pH1Ac-GFP) was excised from the gel and subjected to trypsin digestion. LC-MS/MS analysis of the tryptic fragments and subsequent database searching confirmed that this protein band was the fusion product of Cry1Ac and GFP (Supplementary Table S2). Observation with laser confocal microscope showed that the Cry1AcGFP fusion protein could form large parasporal inclusion body in B. thuringiensis host (Fig. 2). Meanwhile, we found that there existed some protein bands less than 160 kDa for XBU001(pH1Ac-GFP), which were derived from degraded products of the fusion protein as indicated by Western blot analysis using Cry1Ac antibody and GFP antibody (data not shown). Such result might suggest that the fusion protein possessed lower stability and was liable to be degraded while preparing SDS-PAGE sample.
Laser confocal microscopy The synchronously dividing B. thuringiensis cells prepared as described by Bechtel and Bulla (1976) were inoculated into G-Tris medium and cultured at 30 °C with shaking. B. thuringiensis cells cultured for different period were collected and stained with DNA-specific dye Hoechst 33258 (Invitrogen) according to the protocol provided by the manufacturer. Then, the stained cells were observed using laser confocal microscope (Zeiss LSM 710). The localization of DNA and protoxin in B. thuringiensis cells was monitored by observing the blue and green fluorescence signals from Hoechst 33258 a n d G F P. F o r s t a i n i n g w i t h e t h i d iu m b r o m i d e , B. thuringiensis cells were incubated for 30 min at room temperature with ethidium bromide at 1 μg/ml in Tris/acetic acid/EDTA, and then scanned using a laser confocal microscope.
Fig. 1 SDS-PAGE analysis of crystal-spore mixture from sporulated B. thuringiensis strains. Lane M, molecular mass marker; lane 1, recombinant strain XBU001(pH1Ac-GFP); lane 2, strain XBU001(pH1Ac). Cry1Ac-GFP fusion protein and Cry1Ac are indicated by arrows
Appl Microbiol Biotechnol Fig. 2 Distribution of Cry1AcGFP fusion protein and DNA in the recombinant strain XBU001(pH1Ac-GFP) at different growth stages in G-Tris medium. The cells cultured for 5.5, 10.5, 12.5, 14.5, 18.5, and 34 h, respectively, were stained with Hoechst 33258 and observed with a laser confocal microscope. GFP green fluorescent protein signal in the bacterial cells, Hoechst blue fluorescent signal of the Hoechst 33258 dye, DIC differential interference contrast microscopy. The overlay shows overlap of GFP, Hoechst, and DIC graphs. Yellow arrows point to DNA in the cells; red arrows point to inclusion body of Cry1Ac-GFP fusion protein; white arrows point to developing and mature spores. Bar, 2 μm
Solubilization of the parasporal inclusions When the parasporal inclusions from XBU001(pH1Ac-GFP) were pretreated with DNase I and then incubated in 50 mM bicarbonate/carbonate buffer (pH 10.5) containing 2 % (v/v) β-mercaptoethanol, the inclusions were solubilized and the released 160-kDa protein was detected on SDS-PAGE, which was confirmed to be Cry1Ac-GFP fusion protein by in-gel tryptic digestion and LC-MS/MS analysis (Supplementary
Table S2). A DNA band with molecular size of approximate 20 kb could be detected when the solubilized fusion protein was subjected to agarose gel electrophoresis (Supplementary Fig. S2). Distribution of Cry1Ac-GFP and DNA in B. thuringiensis In order to monitor the distribution of protoxin and DNA in B. thuringiensis cells, the recombinant strain
Appl Microbiol Biotechnol
XBU001(pH1Ac-GFP) was cultured in G-Tris medium and analyzed by laser confocal microscopy at different growth stages (Fig. 2). A green fluorescent signal indicated the production and accumulation of the Cry1Ac-GFP fusion protein, while a blue fluorescent signal from Hoechst 33258 indicated the distribution of DNA in the cell. When XBU001(pH1AcFig. 3 Distribution of wild Cry1Ac protoxin and DNA in the recombinant strain XBU001(pH1Ac) at different growth stages in G-Tris medium. The cells cultured for 5.5, 10.5, 14.5, 18.5, and 34 h, respectively, were stained with Hoechst 33258 and observed with a laser confocal microscope. Hoechst blue fluorescent signal of the Hoechst 33258 dye, DIC differential interference contrast microscopy. The overlay shows overlap of Hoechst and DIC graphs. Yellow arrows point to DNA in the cells; red arrows point to crystalline inclusion body of Cry1Ac protoxin; white arrows point to developing and mature spores. Bar, 2 μm
GFP) was grown for 5.5 h, the cells were at the vegetative growth stage. No green fluorescent signal was observed in B. thuringiensis cells, while the DNA was found to be evenly distributed in the cell (yellow arrows). When grown for 10.5 and 12.5 h, GFP signal started to appear in the recombinant cells and generally concentrated as single particle (red
Appl Microbiol Biotechnol
arrows), indicating that the fusion protein was expressed and accumulated as inclusion body. The green fluorescent signal intensity gradually increased with the extension of culture time. Meanwhile, there were two condensed DNA fluorescence signals located in the pole of the cells (yellow arrows). At this stage, no visible spore could be observed. When cultured for 14.5 and 18.5 h, the spore in the recombinant cells could be distinguished (white arrows). DNA signal from the region of spore formation gradually became faint or vanishing with the development of spore, while the other pole or remaining area around the spore and inclusion in the cells still had strong DNA signal. From the very beginning of Cry1Ac-GFP expression to the maturation of inclusion body, it seemed that the DNA fluorescent signal did not appear in the region where the Cry1Ac-GFP inclusion body was assembled. When cultured for 34 h, the spore and inclusion body were released from the lysed cells. The released inclusion body did not exhibit DNA fluorescent signal but part of spores showed very faint DNA signal. Observation of wild Cry1Ac production and DNA distribution in B. thuringiensis As we were concerned that the distribution pattern of Cry1Ac protoxin and DNA in B. thuringiensis cells might be altered owing to the fusion of Cry1Ac and GFP, we further investigated wild Cry1Ac production and DNA distribution in the recombinant strain XBU001(pH1Ac) cultured in G-Tris medium by using DNA-specific dye Hoechst 33258 and laser confocal microscopy (Fig. 3). It was found that there was no obvious difference in growth curve and sporulation efficiency between strains XBU001(pH1Ac-GFP) and XBU001(pH1Ac) (data not shown). When cultured for 5.5 h, the DNA was evenly distributed in the cells. At 10.5 h, there appeared two condensed DNA signals in each pole of the cells (yellow arrows). When cultured for 14.5 and 18.5 h, the spore appeared at one end of the cells (white
Fig. 4 Distribution of wild Cry1Ac protoxin and DNA in the mature unlysed XBU001(pH1Ac) cultured for 18.5 h in G-Tris medium. The cells were stained with ethidium bromide and observed with a laser confocal microscope. EB orange fluorescent signal of the ethidium
arrows) and the bipyramidal crystals of Cry1Ac could be gradually distinguished (red arrows). The spore emitted faint DNA fluorescence while the other end still existed strong DNA signal at 14.5 h. However, the DNA fluorescence in the spore region vanished at 18.5 h and there existed DNA signals in the remaining area of the cells. It was noteworthy that the bipyramidal crystals of Cry1Ac did not exhibit DNA fluorescence. When cultured for 34 h, the spore and crystal were released from the lysed cells and not fluorescent on Hoechst 33258 staining. Furthermore, we treated the sporulated XBU001(pH1Ac) cells with another nucleic acid dye ethidium bromide and visualized them by laser confocal microscopy. It was found that there was no DNA fluorescence from the bipyramidal crystal of Cry1Ac (Fig. 4), which was consistent with the results obtained by Hoechst 33258 staining. The distribution pattern of protoxin and DNA in strain XBU001(pH1Ac) at the sampling time seemed to be similar to that observed in XBU001(pH1Ac-GFP). However, the expression and distribution of Cry1Ac could not be clearly observed before the Cry1Ac protoxins were accumulated as visible inclusion. In contrast, even the small particle formed by Cry1Ac-GFP could be clearly distinguished by GFP signal at the very beginning of expression. For example, the Cry1AcGFP was expressed and accumulated as tiny particle when cultured for 10.5 h, while the expression of wild Cry1Ac could not be monitored in XBU001(pH1Ac) at this time.
Discussion One of the most striking features of B. thuirngiensis is the synthesis of the parasporal crystal during sporulation. The genomic DNA in B. thuringiensis not only direct the formation of spore but also the protoxin synthesis. Especially, it was reported that heterologous 20-kb DNA fragments were intimately associated with the parasporal crystal and they existed
bromide dye, DIC differential interference contrast microscopy. The overlay shows overlap of EB and DIC graphs. Yellow arrows point to DNA in the cells; red arrows point to crystalline inclusion body of Cry1Ac protoxin; white arrows point to spores. Bar, 2 μm
Appl Microbiol Biotechnol
as 20-kb DNA-protoxin complex (Bietlot et al. 1993; Schnepf et al. 1998). In this study, we explored the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages utilizing fluorescent reporter GFP and laser confocal microscopy. The shifting pattern of DNA distribution and the accumulation of Cry1Ac-GFP within the B. thuringiensis cell were clearly presented. Meanwhile, we did not observe co-localization of GFP signal from parasporal inclusion body with DNA fluorescence signal at different growth stages, which conflicted with the previous report that DNA fluorescence appeared in the region where the crystalline inclusion body was assembled in B. thuringiensis (Bietlot et al. 1993). The Cry1Ac-GFP fusion protein was constructed to indicate the cellular localization of Cry1Ac protoxin in this study. As indicated by laser confocal microscopic analysis, the Cry1Ac-GFP fusion protein was effectively expressed in B. thuringiensis host, which might be due to the fact that the transcriptional fusion of cry1Ac-gfp was cloned into the multicopy shuttle vector pHT315 (10 to 15 copies per cell) under the control of the overlapping promoters (BtI and BtII) and the transcription terminator with two stem-loop structures of the cry1Ac gene (Agaisse and Lereclus 1995). These genetic elements could promote transcription of the fusion gene and stability of the produced mRNA. In addition, the deposition of this fusion protein into parasporal inclusion body might decrease its susceptibility to premature proteolytic degradation in B. thuringiensis host. However, the Cry1Ac-GFP fusion protein could not form bipyramidal inclusion as native Cry1Ac, possibly because fusion to GFP interfered with the folding of fusion protein to some extent. Nevertheless, the inclusion body could be solubilized in the alkaline solution and the released fusion protein was associated with DNA. Extensive studies of the sporulation of Bacillus subtilis have provided detailed information on the phenotypes of such differentiation process (Ben-Yehuda et al. 2003; Errington 1993), which was helpful for explaining the distribution pattern of protoxin and DNA in B. thuringiensis. B. subtilis cells that have begun to sporulate contain two condensed chromosomes known as the axial filament, which extends from pole to pole with the origins located at its ends. A septum then forms near one pole and divides the developing cell unequally into a forespore and a mother cell, and then a DNA translocase would pump one chromosome across the septum into the forespore. Our study showed that there existed two strong DNA signals at the pole of the B. thuringiensis cells at the early stages of sporulation, which might correspond to two condensed genomic DNA. One DNA mass would be trapped in the forespore and another would stay in the mother cell. In addition, there existed some space between the two DNA fluorescent regions with nearly round shape. As indicated by our observation, such space seemed to be the region where Cry1Ac-GFP fusion protein was initially deposited as inclusion body. At the subsequent stages of spore formation, the
DNA fluorescence from the region of spore development became faint and ultimately vanished, possibly suggesting that the DNA was gradually enveloped by the developing spore and then totally covered by the mature spore. As to the DNA mass in the mother cell, it gradually became scattered and moved from the pole to the space around the spore and inclusion body with the extension of culture time. During this process, perhaps the produced Cry1Ac-GFP fusion proteins would somehow take up pieces of 20-kb DNA derived from genomic DNA. Interestingly and unexpectedly, we found that there was no DNA fluorescence in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell. Our study using the strain XBU001(pH1Ac) treated with Hoechst 33258 also showed that the DNA fluorescence did not appear in the region where the crystal of Cry1Ac was assembled. However, a previous report (Bietlot et al. 1993) observed a fluorescence signal of DNA from the region of mature crystal in B. thuringiensis subsp. kurstaki HD1 incubated with ethidium bromide under fluorescence microscope. Even though we treated XBU001(pH1Ac) with ethidium bromide, we also did not observe DNA fluorescence from the mature crystal of Cry1Ac using laser confocal microscope. The reason for the discrepancy between the previous report and our results was not known yet. We speculated that the DNA fluorescence previously observed in the region of mature crystal with fluorescence microscope might originate from free DNA surrounding the crystal. The DNA in the growing and mature inclusion body in B. thuringiensis cell could not be seen when staining with DNA-specific dye, possibly because the parasporal inclusion body possessed compact structure and completely covered the embodied DNA, thus preventing access of the fluorescent dye to the DNA. Acknowledgments This research was supported by grants from National Natural Science Foundation of China (30900037), State Key Laboratory of Microbial Technology (M2013-04), Hunan Provincial Natural Science Foundation (12JJ6024, 09JJ3078), and Program for Excellent Talents in Hunan Normal University (ET13105), the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), People’s Republic of China.
References Adang MJ, Staver MJ, Rocheleau TA, Leighton J, Barker RF, Thompson DV (1985) Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensissubsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene 36:289–300 Agaisse H, Lereclus D (1995) How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177:6027–6032 Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119. doi:10.1016/03781119(91)90495-W
Appl Microbiol Biotechnol Bechtel DB, Bulla LA Jr (1976) Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis. J Bacteriol 127:1472–1481 Ben-Yehuda S, Rudner DZ, Losick R (2003) RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299:532–536 Bian X, Huang F, Stewart FA, Xia L, Zhang Y, Müller R (2012) Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering. Chembiochem 13:1946–1952. doi:10.1002/cbic. 201200310 Bietlot HP, Schernthaner JP, Milne RE, Clairmont FR, Bhella RS, Kaplan H (1993) Evidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA. J Biol Chem 268:8240–8245 Chen X, Zaro JL, Shen WC (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65:1357–1369. doi: 10.1016/j.addr.2012.09.039 Clairmont FR, Milne RE, Pham VT, Carrière MB, Kaplan H (1998) Role of DNA in the activation of the Cry1A insecticidal crystal protein from Bacillus thuringiensis. J Biol Chem 273:9292–9296. doi:10. 1074/jbc.273.15.9292 Ellermeier CD, Hobbs EC, Gonzalez-Pastor JE, Losick R (2006) A threeprotein signaling pathway governing immunity to a bacterial cannibalism toxin. Cell 124:549–559. doi:10.1016/j.cell.2005.11.041 Errington J (1993) Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57:1–33 Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Müller R, Stewart AF, Zhang Y (2012) Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol 30:440–446. doi:10.1038/ nbt.2183 Guo S, Li J, Liu Y, Song F, Zhang J (2011) The role of DNA binding with the Cry8Ea1 toxin of Bacillus thuringiensis. FEMS Microbiol Lett 317:203–210. doi:10.1111/j.1574-6968.2011.02230.x Park HW, Ge B, Bauer LS, Federici BA (1998) Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64:3932–3938 Pendleton IR, Morrison RB (1966) Separation of the spores and crystals of Bacillus thuringiensis. Nature 212:728–729
Penfold RJ, Pemberton JM (1992) An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118: 145–146. doi:10.1016/0378-1119(92)90263-O Schernthaner JP, Milne RE, Kaplan H (2002) Characterization of a novel insect digestive DNase with a highly alkaline pH optimum. Insect Biochem Mol Biol 32:255–263. doi:10.1016/S0965-1748(01) 00084-4 Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806 Sun Y, Fu Z, Ding X, Xia L (2008) Evaluating the insecticidal genes and their expressed products in Bacillus thuringiensis strains by combining PCR with mass spectrometry. Appl Environ Microbiol 74:6811– 6813. doi:10.1128/AEM. 01085-08 Sun Y, Wei W, Ding X, Xia L, Yuan Z (2007) Detection of chromosomally located and plasmid-borne genes on 20 kb DNA fragments in parasporal crystals from Bacillus thuringiensis. Arch Microbiol 188: 327–332. doi:10.1007/s00203-007-0252-7 van Frankenhuyzen K (2009) Insecticidal activity of Bacillus thuringiensis crystal proteins. J Invertebr Pathol 101:1–16. doi:10. 1016/j.jip.2009.02.009 Wu F, Zhao X, Sun Y, Li W, Xia L, Ding X, Yin J, Hu S, Yu Z, Tang Y (2012) Construction of gene library of 20 kb DNAs from parasporal crystal in Bacillus thuringiensis strain 4.0718: phylogenetic analysis and molecular docking. Curr Microbiol 64:106–111. doi:10.1007/ s00284-011-0038-7 Xia L, Sun Y, Ding X, Fu Z, Mo X, Zhang H, Yuan Z (2005) Identification of cry-type genes on 20-kb DNA associated with Cry1 crystal proteins from Bacillus thuringiensis. Curr Microbiol 51:53–58. doi:10.1007/s00284-005-4504-y Yin J, Ding X, Xia L, Yu Z, Lv Y, Hu S, Huang S, Cao Z, Xiao X (2011) Transcription of gene in an acrystalliferous strain of Bacillus thuringiensis XBU001 positively regulated by the metalloprotease camelysin gene at the onset of stationary phase. FEMS Microbiol Lett 318:92–100. doi:10.1111/j.1574-6968.2011.02247.x Zhang Y, Muyrers JP, Testa G, Stewart AF (2000) DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol 18:1314–1317. doi:10.1038/82449
