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The gene cyt1Aa is one of the genes in the complex determining the mosquito larvicidity of Bacillus thuringiensis subsp. israelensis. Previous cloning in Escherichia coli resulted in a 48-bp addition upstream, encoding a chimera. Here, cyt1Aa was recloned without the artifact, and its toxicity against Aedes aegypti larvae and host E. coli cells was retested.
Bacillus thuringiensis subsp. israelensis is an effective control agent against mosquito and black fly larvae (13). Its larvicidal activity is determined by four major polypeptides of the parasporal crystalline body produced during sporulation: Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa. The encoding genes are mapped on the 128-kb plasmid pBtoxis (3). Cyt1Aa has low toxicity and specificity but is the most synergistic to any of the other three (9) and hence has a crucial role in suppressing resistance to Cry toxins in the targets (19). In addition, Cyt1Aa exerts, a general cytolytic effect on a variety of prokaryotic (5, 20) and eukaryotic (16) cells by a detergent-like mode of action (12). This property makes it potentially useful for treating bacterial infections and even cancerous growths if specificity to tumor cells is raised by genetically fusing Cyt1Aa to targeting ligands (6).
cyt1Aa was cloned in Escherichia coli, for the first time, as part of a 10-kb HindIII digestion fragment of pBtoxis on pUC12 (18) and its sequence (GenBank accession number X03182) was determined (17). Attempts to subclone it separately under its own promoter had failed due to the antibacterial activity of Cyt1Aa (1, 8), unless coproduced with a 20-kDa protein encoded by p20 that is carried on the same HindIII fragment (1, 10). Cloning cyt1Aa alone had been achieved only under more-stringent repression conditions, by placing it under the promoter recognized by phage T7 RNA polymerase (8), encoded by the gene that was induced by isopropyl-β-d-thiogalactopyranoside (IPTG) from the chromosome of E. coli strain BL21 (15). However, this expression system as such inhibits cell metabolism; hence, investigating the in vivo effect of Cyt1Aa on E. coli requires a lenient system. The current procedure to engineer restriction sites into PCR primers enables one to easily clone cyt1Aa under any desired promoter. To achieve this purpose, we exploited vector pUHE-24S (kindly provided by Stefan Leu), a modified pUHE-24 (University of Heidelberg, Germany) from which one NcoI site had been removed, leaving a unique NcoI site in the translation start codon. pUHE-24 is a descendant of pDS (7) containing a T5 ribosome-binding site (4), two tandem lacO1, and the early T7 promoter PA1 (utilizing the usual E. coli RNA polymerase) (Fig. (Fig.1A1A).
The HindIII site in the vector multicloning site (MCS), downstream of the desired NcoI site, was used to clone cyt1Aa because its reading frame contains an NcoI restriction site 46 nucleotides from the translation start codon ATG. The corresponding HindIII site in the cyt1Aa insert was PCR generated in the upstream region of the gene (Fig. (Fig.1B).1B). The created clone expressed cyt1Aa upon IPTG induction and was successfully used for the subsequent research (10, 11).
However, it was recently determined that this upstream region of cyt1Aa along with the rest of the MCS of pUHE-24S was included in-frame with cyt1Aa and the vector ATG, extending the gene by 48 nucleotides at its 5′ end, and was thus translated into a Cyt1Aa version with 16 additional amino acids at its N terminus (Fig. (Fig.1B).1B). Since this N-terminal tail may adversely affect stability/activity of the protein, it was decided to reclone the native gene without that artifact.
To achieve this end, the cyt1Aa intrinsic NcoI site was eliminated by Stratagene's QuikChange site-directed mutagenesis kit. Plasmid pRM4-C (10) was used as a template to produce two adjacent point mutations, eliminating the NcoI restriction site (shown in boldface type) (Fig. (Fig.1C)1C) and creating an EcoRV restriction site (underlined), together with the following primers: 5′-GTCCATTAGAAGATATCAAGGTAAATCCCTGGAAAACCCCTC-3′ and 5′-GAGGGGTTTTCCAGGGATTTACCTTGATATCTTCTAATGGAC-3′.
Replacement of nucleotides for the above mutations does not change the corresponding amino acids (compare Fig. 1B and C). The mutations in the generated plasmid [pRM4-C(Mut)] were confirmed by digestion with the restriction endonuclease and by DNA sequence analysis.
cyt1Aa from pRM4-C(Mut) was amplified with Vent polymerase using the following primer pair: 5′-GTTTATCCATGGAAAATTTAAATCATTGTCC-3′ and 5′-CGCGAGGCAGCTCTAGATTAACGC-3′. The PCR products were digested with NcoI and XbaI and inserted into the respective sites of pUHE-24S, generating pRM4-C(Nat).
Transgenic E. coli, grown at 37°C in ampicillin-containing LB medium, was induced by IPTG (0.5 mM) at an optical density (at 660 nm) of 0.1 to 0.2 (about 2 × 107 cells ml−1). Cells were harvested by centrifugation before induction and, after 1, 2, and 4 h, were resuspended in water, and aliquots were boiled (10 min) in a sample treatment buffer. Proteins were separated by SDS-PAGE on 13% gel, electrotransferred onto nitrocellulose membranes, and exposed to antiserum directed against Cyt1Aa (kindly provided by Sarjeet Gill, University of California, Riverside). Alkaline phosphatase-conjugated protein A was used as a detector, and the antigen was visualized with chromogenic substrate for alkaline phosphatase.
The immunoblot (Fig. (Fig.2)2) clearly demonstrates one band of ca. 25 kDa produced from pRM4-C(Nat), while the corresponding band representing Cyt1Aa produced from pRM4-C is slightly larger. This is consistent with the suggestion that the initial cloning of cyt1Aa created an extended product upon expression. This conclusion was validated by sequencing the respective genes.
The clones pRM4-C and pRM4-C(Nat) were compared regarding their toxicity to E. coli (Fig. (Fig.3)3) and synergy to the cry4Aa-expressing clone against Aedes aegypti larvae (Fig. (Fig.4).4). For these purposes, IPTG-induced clones were tested, respectively, for colony-forming ability (after 24 h of incubation at 37°C) on LB plates with 100 μg ampicillin ml−1 and for killing early-third-instar A. aegypti larvae 24 h (at 28°C) after being mixed with the cry4Aa-expressing clone in disposable cups with 100 ml sterile tap water. No significant difference was found between the clones, concluding that the additional 16 amino acids do not significantly affect Cyt1Aa activities. Viable counts of induced cells appear to recover (Fig. (Fig.3B),3B), most likely due to spontaneous mutants that gained resistance to the lethal effect of Cyt1Aa (11).
Translation of noncoding DNA sequences in-frame with cloned genes can potentially present an obstacle for the study of the corresponding protein, and special attention must be paid to the cloning procedure so that the desired gene product is obtained. The specific modification described here did not change the activity of Cyt1Aa produced in E. coli. The unaffected larvicidity against A. aegypti is simply explainable since Cyt1Aa undergoes activation by proteolysis in the larval gut; a fragment of about 30 amino acids is cleaved off the N terminus of native Cyt1Aa during processing (2), obviously including the 16 amino acids previously added inadvertently. On the other hand, the unaffected bactericidal level against its host E. coli implies that the above-described modification is probably too small to impair proper folding for antibacterial activity of Cyt1Aa. This fact can be used to engineer genetically modified versions of Cyt1Aa fused to short ligand-peptides that target its cytolytic activity to specific cell types as desired (6).
Thanks are due to Monica Einav for providing technical assistance.
This investigation was partially supported by grant no. 2007-037 of the United States-Israel Binational Science Foundation, Jerusalem, Israel.
Published ahead of print on 26 March 2010.