New Variants of Lepidoptericidal Toxin Genes Encoding <b><i>Bacillus thuringiensis</i></b> Vip3Aa Proteins

Keywords: 

subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP. 

Introduction

The entomopathogenic bacteria Bacillus thuringiensis is characterized by its ability to produce parasporal crystalline inclusions during sporulation containing insecticidal proteins such as Cry and Cyt proteins [Sauka and Benintende, 2008]. In addition, many B. thuringiensis strains are known to produce other kinds of insecticidal proteins during the vegetative growing phase that do not form crystals which are called Vip (vegetative insecticidal proteins) [Estruch et al., 1996]. This type of proteins includes Vip1, Vip2 and Vip3. Vip1 and Vip2 are the components of binary toxins that have coleopteran specificity, whereas Vip3 proteins have activity against a wide variety of lepidopteran pests [Estruch et al., 1996;Warren, 1997]. To date, these three groups of Vip proteins can also be classified into 9 subgroups, 27 classes and 92 subclass-es according to their amino acid sequence similarity (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/ Bt/vip.html). 
Among these proteins, Vip3A do not share homology with other known proteins and act against lepidopteran larvae through a unique mechanism of action [Bhalla et al., 2005;Estruch et al., 1996;Lee et al., 2003Lee et al., , 2006]]. These proteins act by binding to specific receptors, different when compared to that of Cry, located in the midgut epithelium of susceptible insect species. Then, cation-specific pores are formed that disrupt ion flow in the midgut, causing paralysis and death [Lee et al., 2006]. All these factors emphasized the possible benefit of use of Vip3A in resistance management strategies against Cry proteins, since novel vip3A genes that are discovered from B. thuringiensis strains could be useful as biological control agent against resistant or insensitive pests [Liu et al., 2007]. 
Some polymerase chain reaction (PCR)-based methods have been developed to detect vip3A genes from B. thuringiensis strains [Asokan et al., 2012;Beard et al., 2008;Bhalla et al., 2005;Hernández-Rodriguez et al., 2009;Liu et al., 2007;Rice, 1999;Yu et al., 2011]. The present study reported a new PCR-restriction fragment length polymorphism (RFLP) method that can distinguish between known and novel vip3A genes from B. thuringiensis strains. Furthermore, 4 novel vip3Aa genes were cloned and sequenced. 

Results and Discussion

In order to identify genes within the vip3A gene family, a new strategy based on PCR-RFLP was used. The specific primers used for PCR hybridize in highly conserved sequences flanking a highly variable region of vip3A genes. These produced amplification in 12 B. thu ringiensis strains ( fig. 1 ). B. thuringiensis serovar kurstaki strains HD-1 and HD-73 were used as positive and negative controls, respectively. Polyacrylamide gel data confirmed that 10 strains had a vip3Aa -type RFLP pattern, with 3 main bands of 240, 233, and 111 bp when the product was digested with Dde I conforming to the predicted fragments of vip3Aa genes. Two vip3Ab -type RFLP patterns were identified in B. thuringiensis serovar sumiyoshiensis 4AO1 and japonensis 4AT1, showing 3 main bands of 233, 159 and 111/105 bp. Moreover, a novel vip3A -type pattern with 2 bands of 344 and 264 bp was identified in B. thuringiensis serovar entomocidus HD-110. Representative restriction patterns of vip3A genes are shown in figure 1 . 
Afterwards, vip3A genes of B. thuringiensis serovar kurstaki HD-1, entomocidus HD-110, sumiyoshiensis 4AO1 and japonensis 4AT1 were chosen for cloning and sequencing by using specific and vector primers ( table 1 ). To our knowledge, there are no previously published studies concerning vip genes with a full description of all the primers needed for sequencing. The oligonucleotide primers, SRF and SRRI, were successfully used for the amplification of the full length of vip3A genes. All the obtained sequences have in common that they were 2,370 nucleotides long, encoding 790 amino acid residues. The genes from these strains were named vip3Aa33, vip3Aa37, vip3Aa40 and vip3Aa41, respectively, by the Bt Pesticidal Crystal Protein Nomenclature Committee (http://www. lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). 
The nucleotide sequence of the gene vip3Aa33 from serovar kurstaki HD-1 strain was identical to vip3Aa7, 9, 10, 12, 15 and 21 . A substitution at position 891 of C for G (transversion) was found to be present in the almost identical gene vip3Aa37 from serovar entomocidus HD-110 strain. This point mutation constituted a silent mutation that did not result in amino acid substitutions, but generated a new Dde I restriction site. Consequently, this new restriction site produced a novel RFLP pattern totally different to that of the other vip3A genes ( table 2 ). On the other hand, nucleotide sequences of vip3A genes from serovar japonensis 4AT1 and sumiyoshiensis 4AO1 strains showed to be the most different in comparison to other vip3Aa genes. They seem to be more similar to vip3Ab, d, e, f, g and h, and sequence data revealed that they were not vip3Ab genes either as suggested by Dde I restriction analysis. The restriction analysis for identification of vip3A genes was originally designed as a 2-step process. As was vip3Aa,vip3Ab,vip3Ae,vip3Af,vip3Ad,vip3Ah 3A2F,reg 5 GCT AAG CTA AAA C AAA ATT ATC AAG 49.3 1124-1147 vip3Aa, vip3Ab, vip3Ae, vip3Af, vip3Ad, vip3Ah 3A2R, reg 3 ATT TGT TCA GAT TGA TCT GGR CA 53.6 1201-1223 vip3Aa, vip3Ab, vip3Ae, vip3Af, vip3Ad, vip3Ah 3A3FI, reg 260, 198, 77, 58, 58, 15 190, 159, 77, 70, 58, 39, 15 Vip3 proteins, and showed the highest sequence similarity shared with Vip3Aa41 (99%). They differed at 5 amino acid residues: Met (3) for Lys, Leu ( 16) for Phe, Leu (244) for Phe, Ser (584) for Phe and Tyr (695) for Asn. Both sequences shared high similarity with Vip3Aa19 (97 and 96%, respectively). There were different amino acid residues between these 3 deduced proteins ( table 4 ). Some previous reports have shown that changes in amino acid residues in Vip3A proteins could affect their insecticidal activity [Doss et al., 2002;Estruch et al., 1996;Liu et al., 2007;Selvapandiyan et al., 2001]. Some of the differences in 19 residues between Vip3Aa19 and Vip3Aa11 were important for lack of toxicity of Vip3Aa19 against Plutella xylostella [Liu et al., 2007]. Moreover, differences in 2 residues between Vip3Aa9 and Vip3Aa1 proteins, and in only 1 residue between Vip3Aa9 and Vip3Aa10 proteins, showed to be involved in the ineffectiveness of Vip3Aa9 protein against Agrotis ipsilon [Doss et al., 2002;Estruch et al., 1996;Selvapandiyan et al., 2001]. Further studies are needed to express vip3Aa40 and vip3Aa41 genes, and to determine toxicity of their products against certain lepidopteran pest that have shown to be insensitive or poorly sensitive to other known Vip3A proteins. 
vip3Aa vip3Aa vip3Aa vip3Aa - - - - - - - - - - - vip3Aa37 - - - - vip3Aa vip3Aa - - - - - - - - - - vip3Aa40 - - - vip3Aa - - - - - vip3Aa 
- - - vip3Aa - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - vip3Aa41 - - vip3Aa - - - - -=
The vip3A gene content of B. thuringiensis strains is listed in table 3 . We did not find any strain that harbored vip3Ab, vip3Ad,vip3Ae,vip3Af,vip3Ag,vip3Ah or combinations between any one of this class of genes. 
It is known that vip3Aa genes are widely distributed among B. thuringiensis [Bhalla et al., 2005;Hernández-Rodriguez et al., 2009;Yu et al., 2011]. To the best of our knowledge, just one study has reported the detection of vip3A genes of strains representing different serovars of standard B. thuringiensis [Bhalla et al., 2005]. These in-vestigators analyzed 24 B. thuringiensis strains obtained from Bacillus Genetic Stock Center (USA). 
The B. thuringiensis serovar thompsoni HD-542 was reported to lack a vip gene [Bhalla et al., 2005], but here we showed that it harbors a vip3Aa gene. This discrepancy could be due to the primers used in that study, which were designed based on the nucleotide sequence of vip3Aa1 only and did not recognize the vip3Aa gene of this strain. In contrast, the serovar pakistani HD-395 (= 4P1) strain was reported to contain a vip3A gene [Bhalla et al., 2005], but here we could not confirm the presence of this class of genes in this strain. 
We identified vip3Aa genes in serovar kurstaki HD-1, alesti HD-4 and wuhanensis HD-525 strains. In that previous work, these B. thuringiensis strains produced PCR products that detected a vip3 gene [Bhalla et al., 2005]. 
A strain from the mosquitocidal serovar israelensis was reported to contain a vip3A gene [Bhalla et al., 2005], but here we showed the lack of this class of genes in strains belonging to this serovar and in others with recognized toxicity for mosquitoes such as kyushuensis, medellin, morrisoni, neoleonensis and jegathesan serovars. 
The differences between Vip3Aa40 and 41 and the Vip3A proteins are spread throughout the sequence, but the C-terminal end of Vip3A40 and 41 is highly conserved in comparison with the other Vip3Aa proteins and has been reported to be crucial for resistance to proteases [Estruch et al., 1996]. On the other hand, the N-terminal end of Vip3A40 and 41 is highly conserved, but in comparison with other non-Vip3Aa-type proteins. 
The N-terminus of the putative signal sequence of Vip3Aa40 and 41 is, with the exception of some amino acids, identical to that of Vip3A toxins except for other 
K S F F H M S K E N G E I R F Vip3Aa41 M S L L H M S K E N G E I R S Vip3Aa19 K N F F Y T F N G S E D K K F
N E T V I R N G N N T I L L L Vip3Aa41 N E T V I R Y G N N T I G G L Vip3Aa19 D K I T T S N R I Y S M F E I
Vip3Aa and Vip3Ah sequences ( fig. 2 ). Although different, these sequences have the high asparagine content that characterizes the N-terminal end of signal sequences in Bacillus [Pugsley, 1993;Rang et al., 2005]. The last 17 amino acids of the signal sequence are identical in all Vip3A sequences ( fig. 2 ). The complete putative signal sequences of Vip3Aa40 and 41 are therefore MNKNNT-KLSARALPSFIDYFNGIYGFATGIKDI and MNMNNT-KLSARALPSLIDYFNGIYGFATGIKDI, respectively. It is known that Vip3Aa proteins are processed at two lysine-rich sites [Estruch et al., 1996[Estruch et al., , 1998]. The first processing site is located in Vip3Aa at Lys 198 in the sequence 192-SSKVKK-199 ( fig. 2 ). This region is significantly different in Vip3Aa40 and 41, in which it is replaced by the sequence 192-TLKVKK-199 being also highly similar to other non-Vip3Aa-type proteins ( fig. 2 ). The 66-kDa insecticidal moiety of Vip3Aa starts at Gly 200 and contains the sequence 199-GSPADIL-207 [Estruch et al., 1998]. The corresponding region in Vip3Aa40 and 41 proteins is 199-SSPADIL-207 ( fig. 2 ). The substitution of Ser (200) for Gly is also conserved in other non-Vip3Aa-type proteins. The second processing site in Vip3Aa is located at amino acid 455 and results in the release of a 33-kDa fragment ranging from residue 200 to residue 455 [Estruch et al., 1998]. In Vip3Aa proteins, the sequence of this second processing site is DLNKKKVESS, DLNKTKVESS or DLNKKLVKSS ( fig. 2 ). In this case, Vip3Aa40 and 41 contains the sequence DLNKKKVESS which is present in most of the Vip3A proteins. 
Vip3Aa40 and 41 contain the same 3 Cys residues in the proteins as most of the Vip3A proteins, except Vip3Aa14 (just 2) and Vip3Ad, g and h (the 3 Cys plus another at the C-terminus). It is likely that some of these cysteines are involved in disulfide bridges and may play a role in the stability of Vip3A proteins. 
PCR-RFLP is important to identify the existence of known insecticidal genes and detect novel ones in B. thuringiensis strains. Novel insecticidal genes were dis- covered by this method, e.g. the novel cry1Ie1 from B. thuringiensis isolate Btc007 [Song et al., 2003]. Even though a PCR-RFLP method can be considered old, as shown through the course of this study, we conclude that it is still useful for the identification of novel genes. This is the first study where variants of the same subclass of insecticidal genes could be distinguished. As seen in table 2 , vip3Aa37, vip3Aa40-41 and the rest of vip3Aa genes could be differentiated using Dde I restriction analysis. 

Experimental Procedures


B. thuringiensis Strains

Eighty-seven B. thuringiensis strains were kindly provided by different institutions around the world ( table 3 ). 

Detection of vip3A Genes

Novel primers for the specific detection of vip3A genes were designed following a previously described methodology [Sauka et al., 2006[Sauka et al., , 2007]]. Primers used for amplification of a vip3Ab,vip3Ad,vip3Ae,vip3Af,vip1Ag and vip1Ah genes were as follows: DS3AF (forward; 5 -GTG AAA ACA AGT GGC AGT G-3 ) and DS3AR (reverse; 5 -TCC GCT TCA CTT GAT TCT ACT-3 ). 
The DNA templates for PCR were obtained as previously described [Sauka et al., 2006]. Five microliters of supernatant was used in each reaction. All the reactions were performed with a final volume of 25 l containing final concentrations of 50 m M KCl, 2.0 m M MgCl 2 , 10 m M Tris-HCl (pH 8.3), 200 M of each deoxynucleoside triphosphate (dATP, dTTP, dGTP, and dCTP), 16 pmol of each primer, and 2.5 U of Taq polymerase (Invitrogen). The PCR amplification consisted of DNA denaturation at 94 ° C for 2 min followed by 25 cycles of amplification with a thermocycler (Eppendorf Mastercycler gradient). Each cycle consisted of a denaturation step at 94 ° C for 1 min, an annealing step at 49 ° C for 45 s, and a chain elongation step at 72 ° C for 45 s. The final elongation step was extended for an additional 5 min. Finally, 10 l PCR product was analyzed by 1.5% agarose gel electrophoresis. 

Identification of vip3A Genes

For the identification of different vip3A genes, 10 l of positive PCR product was digested with 2.5 U Dde I according to manufacturer's instructions (Promega, Madison, Wisc., USA), analyzed by 12% polyacrylamide gel electrophoresis and stained with ethidium bromide. If necessary, samples were digested with 2.5 U Fok I or Alu I. Expected restriction fragment sizes of known and novel vip3A genes were determined by in silico digestion of their available sequences on the B. thuringiensis toxin nomenclature website (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/ Bt/) with the software 'RestrictionMapper' (http://www.restrictionmapper.org/) ( table 2 ). 
Amplification, Cloning and Nucleotide Sequencing of Full-Length vip3A Genes Primers used for the amplification of the whole open reading of vip3A genes ( table 1 ) were designed following a previously described methodology [Sauka et al., 2006[Sauka et al., , 2007]]. The forward primer is class specific and was designed to begin at the ATG initiation codon. The reverse primers were designed from sequences at the 3 end of all vip3A genes. Manual 'hot start' PCR was performed with a final volume of 50 l containing 50 m M KCl, 20 m M Tris-HCl (pH 8.4), 200 m M of each deoxynucleoside triphosphate (dATP, dTTP, dGTP, and dCTP), 1 m M of each primer and 2 m M MgCl 2 . The PCR amplification consisted of an initial denaturation step of 2 min at 94 ° C, followed by 29 cycles of 5 s at 95 ° C, 20 s at 47 ° C, 4 min at 68 ° C and a final elongation step of 5 min at 68 ° C in a thermocycler (Eppendorf Mastercycler gradient). Five units of Taq DNA polymerase (Invitrogen) were added after the first denaturation step. Finally, the PCR product was analyzed by 1.0% agarose gel electrophoresis stained with ethidium bromide. The PCR product of 4 B. thuringiensis strains were purified from the agarose gel matrix using Wizard SV Gel and PCR Clean-Up System (Promega), cloned in pGEM-T Easy vector (Promega) and then transformed into competent Escherichia coli JM109 strain following the manufacturer protocols. Fifteen white colonies were selected on X-gal IPTG containing selective LB agar plates. Verifying whether the clones contained inserts was accomplished by PCR of recombinant plasmid DNA using vector primer SP6 and T7. Afterwards, the vip3A gene of each clone was identified by the PCR-RFLP method described above. Three clones harboring vip3A genes were sequenced in both directions by primer walking using vector (SP6 and T7) and specific primers detailed in table 1 in the 'Unidad de Genómica' (INTA, Argentina). 

Sequence Comparisons

These vip3A nucleotide sequences and their translations into amino acid sequences (with the ExPASy translate tool; http:// www.expasy.org/tools/dna.html) were aligned separately with ClustalW [Thompson et al., 1994]. The deposited sequences in GenBank of all vip3A genes were accessed through the B. thuringiensis toxin nomenclature website and used as references. 

Nucleotide Sequence Accession Numbers

The DNA nucleotide sequences of vip3A genes from B. thuringiensis serovar entomocidus HD-110, japonensis 4AT1, sumiyoshiensis 4AO1 and kurstaki HD-1 have been deposited in the Gen-Bank databases (http://www.ncbi.nlm.nih.gov) under the accession numbers HM132041, HM132042, HM132043 and GU073128, respectively. 



  Fig. 1. a PCR amplification with oligonucleotide primers DS3AF and DS3AR of vip3A genes in B. thuringiensis strains. Lane 1: HD-133; lane 2: HD-137; lane 3: T07001; lane 4: HD-4; lane 5: HD-110; lane 6: T05001; lane 7: HD-155; lane 8: 4AT1; lane 9: HD-542; lane 10: HD-125; lane 11: 4AO1; lane 12: 4T1; lanes 13-14: HD-1 and HD-73 used as positive and negative controls, respectively. b Representative PCR-RFLP patterns of vip3Aa, vip3Ab / vip3Aa40-41 and vip3Aa37 -type genes digested with Dde I (lanes 1, 2 and 3, respectively), and vip3Aa40-41 -type genes digested with Fok I (lane 4). MW = Molecular weight marker with sizes indicated on the left (bp). 


Fig. 2 .

 2 Fig. 2. Alignment of the Vip3Aa40 and 41 sequences with the sequence of a representative of each subclass of Vip3A toxins. Asterisks below the sequences indicate conserved amino acids. The black boxes indicate regions described in the text. The black box labeled S indicates the signal peptide sequence, the one labeled P indicates the proteolytic processing sites characterized in Vip3Aa and the box labeled I indicates the beginning of the 66-kDa insecticidal fragment characterized in Vip3Aa. 


Table 1 .

 1 P rimers for sequencing of vip3A genes 


	vip3A primers a Sequence b	Tm, °Cc Position d	Gene/s recognized
	SRF, reg 5	ATG AAC AWG AAT AAT ACT AAA TTA ARC RCA 52.4	1-30	vip3Aa, vip3Ab, vip3Ad, vip3Ae, vip3Af, vip3Ag, vip3Ah
	SRR I, reg 3'	TTA CTT AAT AGA GAC ATC GTA AAA ATG TAC 51.8	2341-2370 vip3Aa
	SRR II, reg 3	TTA CTT AAT TGA AAA ATC TCG GAA AKT TAT 51.7	2335-2364 vip3Ab, vip3Ae, vip3Ag
	SRR III, reg 3 TTA TTT AAT AGA AAC GTT TTC AAA TGA TAT	49.6	2338-2367 vip3Af
	SRR IV, reg 3 TTA TTT AAT AGA RAA ATY ATA AAA RTR TRC	47.4	2332-2361	vip3Ad
				2359-2388	vip3Ah
	3A1F, reg 5	ACT GAG TTA ACT GAA CTA GCG	52.0	627-648	vip3Aa, vip3Ad, vip3Ah
	3A1R, reg 3	GCT GAA CGC CCG AAT AAA TTA	53.4	723-741	

Table 2 .

 2 
	The nucleotide sequence of vip3Aa40 from serovar
	japonensis 4AT1 and vip3Aa41 from serovar sumiyo-
	shiensis 4AO1 shared the highest similarity (98 and
	97%) with the vip3Aa19 (DQ241674) gene. The deduced
	Vip3Aa40 protein sequence was compared with other

E xpected restriction fragment sizes of digested vip3A genes shown, the PCR product is first digested with Dde I and if necessary with Alu I ( table 2 ). Based on these sequences and using in silico restriction analysis with Fok I as a second step first, we could experimentally differentiate later vip3Aa40-41 genes from vip3Ab genes. 
Table 3 .

 3 T he vip3A gene content of exotic B. thuringiensis strains 


	B. thuringiensis strain	Source	Gene subclass	B. thuringiensis strain	Source	Gene subclass
	aizawai HD-133	USDA				
	aizawai HD-137	USDA				
	aizawai T07001	Pasteur				
	alesti HD-4	USDA				
	argentinensis 4BV1	BGSC				
	asturiensis 4BQ1	BGSC				
	azorensis 4CB1	BGSC				
	balearica 4BK1	BGSC				
	cameroun 4AF1	BGSC				
	canadensis 4H2	BGSC				
	chanpaisis 4BH1	BGSC				
	colmeri 4X1	BGSC				
	dakota 4R1	BGSC				
	darmstadiensis 4M1	BGSC				
	darmstadiensis 4M2	BGSC				
	entomocidus HD-110	USDA				
	entomocidus 4I1	BGSC				
	entomocidus 4I3	BGSC				
	finitimus HD-3	USDA				
	fukuokaensis 4AP1	BGSC				
	galleriae T05001	Pasteur				
	galleriae HD-155	USDA				
	graciosensis 4CD1	BGSC				
	higo 4AU1	BGSC				
	huazhongensis 4BD1	BGSC				
	iberica 4BW1	BGSC				
	indiana 4S2	BGSC				
	israelensis HD-500	USDA				
	israelensis HD-567	USDA				
	israelensis HD-522	UNAM				
	israelensis IPS-82	CINVESTAV				
	israelensis T014001	Pasteur				
	japonensis 4AT1	BGSC				
	jegathesan B-23141	BGSC				
	jinghongiensis 4AR1	BGSC				
	tenebrionis DSM2803	CINVESTAV				
	thompsoni HD-542	USDA				
	thuringiensis HD-2	USDA				
	thuringiensis T01001	Pasteur				
	thuringiensis T01026	Pasteur				
	tochigiensis 4Y1	BGSC				
	tohukensis 4V1	BGSC				
	tolworthi HD-125	USDA				
	toumanoffi 4N1	BGSC				



  No positive PCR signal obtained with primers DS3AF/DS3AR. from http://karger.com/mmb/article-pdf/22/6/373/3098711/000345911.pdf by Indiana University Libraries user on 30 August 2023 


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Table 4 .

 4 T he differences of amino acid residues among Vip3Aa40 and 41 compared to Vip3Aa19 


	Protein	A mino acid residue											
		3	9	16	244	355	396	442	470	498	543	546	547	553	571	584
	Vip3Aa40														

JMol Microbiol Biotechnol 2012;22:373-380 

Downloaded from http://karger.com/mmb/article-pdf/22/6/373/3098711/000345911.pdf by Indiana University Libraries user on 30 August 2023 

Downloaded from http://karger.com/mmb/article-pdf/22/6/373/3098711/000345911.pdf by Indiana University Libraries user on 30 August 2023 Sauka/Rodriguez/Benintende J Mol Microbiol Biotechnol 2012;22:373-380 

Acknowledgements

Diego H. Sauka holds a research career award from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
Diego H Sauka, Sonia E Rodriguez, Graciela B Benintende
New Variants of Lepidoptericidal Toxin Genes Encoding <b><i>Bacillus thuringiensis</i></b> Vip3Aa Proteins