Interspecies Horizontal Transfer and Specific Integration of the Mosquitocidal Toxin-Encoding Plasmid pTAND672-2 from Bacillus thuringiensis subsp. israelensis to Lysinibacillus sphaericus

Abstract

pTAND672-2, a 144-kb resident plasmid of Bacillus thuringiensis serovar israelensis strain TAND672, was sequenced and characterized. This extrachromosomal element carries mosquitocidal toxin-, conjugation-, and recombinase-encoding genes, together with a putative arbitrium system, a genetic module recently discovered in temperate phages controlling lysogeny-lysis transition and in mobile genetic elements (MGEs) where its function remains clarified. Using conjugation experiments, pTAND672-2 is shown to be a novel integrative and conjugative element (ICE), which can horizontally transfer from B. thuringiensis serovar israelensis to Lysinibacillus sphaericus, another mosquitocidal bacterium, where it integrates into the chromosome. Its integration and circularization are reversible and involve a single-cross recombination between 33-bp specific sites, attB in the chromosome of L. sphaericus and attP in pTAND672-2. CDS143, coding for the putative tyrosine integrase Int143 distantly related to site-specific tyrosine Xer recombinases and phage integrases, can mediate the integration of pTAND672-2 to attB. The B. thuringiensis mosquito-killing genes carried by pTAND672-2 are efficiently transcribed and expressed in L. sphaericus, displaying a slight increased toxicity in this bacterium against Aedes albopictus larvae. The occurrence of pTAND672-2-like plasmids within the Bacillus cereus group was also explored and indicated that they all share a similar genetic backbone with diverse plasmid sizes, ranging from 58 to 225 kb. Interestingly, among them, the pEFR-4-4 plasmid of Bacillus paranthracis EFR-4 and p5 of B. thuringiensis BT-59 also display conjugative capability; moreover, like pTAND672-2 displays a chimeric structure between the pCH_133-e- and pBtoxis-like plasmids, pBTHD789-3 also appears to be mosaic of two plasmids. IMPORTANCE Horizontal transfer of mobile genetic elements carrying mosquitocidal toxin genes may play a driving role in the diversity of mosquitocidal bacteria. Here, the 144-kb mosquitocidal toxin-encoding plasmid pTAND672-2 is the first verified integrative and conjugative element (ICE) identified in Bacillus thuringiensis serovar israelensis. The key tyrosine integrase Int143, involved in the specific integration, is distantly related to other tyrosine recombinases. The study also reports the occurrence and potential interspecies transmission of pTAND672-2-like plasmids with varied sizes in B. thuringiensis, Bacillus paranthracis, and Bacillus wiedmannii isolates belonging to the Bacillus cereus group. This study is important for further understanding the evolution and ecology of mosquitocidal bacteria, as well as for providing new direction for the genetic engineering of biopesticides in the control of disease-transmitting mosquitoes.

Keywords: Bacillus thuringiensis serovar israelensis; Lysinibacillus sphaericus; conjugation; integrative and conjugative element; mobile genetic elements; mosquitocidal toxin; tyrosine recombinase.

FIG 1

Circular map comparison among pTAND672-2, pCH_133-e, and pBtoxis. pCH_133-e and pBtoxis display 77% and 43% coverage to pTAND672-2, respectively, with 100% identity. No plasmid replication/partition genes were predicted in the pBtoxis-like region in pTAND672-2. The overlap regions among the three plasmids contain several IS elements.

FIG 2

Schematic diagram of pTAND672-2 integration and excision within the transconjugant. Black, white, and gray blocks indicate the location of 33-bp specific recognition sites attP/attL and attB/attR in donor, recipient, and transconjugant, respectively, in which the white one indicates the identical half-sites between attP and attB or attL and attR. Thin gray and green arrows showed the primer sets INT-a, INT-b, INT-c, and INT-d designed according to the flanking sequences of attB and attP for testing integration and excision of pTAND672-2. Six nucleotide variants in the attP/attL and attB/attR are shown in italic and underlined. The gene of the tyrosine integrase (CDS143) containing the DNA_BRE_C domain, neighboring the insertion site in pTAND672-2, is marked with a black star (in the chromosome) (bottom) or with a dotted blue rectangle (in pTAND672-2) (top). Three phage-associated genes downstream the integration insertion site on the chromosome (Bsph_1945, Bsph_1947, and Bsph_1949) are marked as light blue arrows.

FIG 3

PCR verification of the transconjugant BS-pTAND672-2 and of pTAND672-2 integration, excision, or circularization under various growth conditions. (A) PCR products obtained using donor (D), recipient (R), and transconjugants (TsS, T3, T8, T9, T11) as templates for amplifying toxin-encoding genes cry4Aa, cry10Aa, and bin, respectively. TsS was sent for genome sequencing to obtain the complete sequence (GenBank accession number CP071738). M, D2000 plus DNA marker. (B) PCR detection of pTAND672-2 integration, excision, or circularization within BS-pTAND672-2 picked from different colonies. M, D2000 plus DNA marker. The donor TAND672 and the recipient G725Δ0498 were set as controls. (C) Sequencing verification of the integration and circularization capability. PCR products containing attL, attR, attP, and attB sites were sequenced to verify if intact attB in the chromosome of the host is interrupted by linearized pTAND672-2 from the donor TAND672. Brown and purple arrows indicate the sequences from the recipient chromosome and pTAND672-2, respectively. (D) The influence of the medium and growth phases on excision and circularization efficiency. M, Trans2k PlusII DNA marker; CK, negative control using double-distilled water (ddH₂O) as the template. (E) The influence of the synthesized AimP peptide (YMVDPGGMG) at final concentrations of 0, 1, and 2.5 μM in LB medium and different growth phases (4, 6, and 24 h) on excision and circularization efficiency of pTAND672-2 in the transconjugant BS-pTAND672-2. M, D2000 plus DNA marker; CK D and R indicate the donor TAND672 and the recipient G725Δ0498 setting as controls, respectively. (a and b) Detection of the integration by the occurrence of attL and attR, respectively. (c) Detection of the circularization by the occurrence of attP. (d) Detection of the excision or nonintegration by the occurrence of attB. (e) Detection of the housekeeping gene ccpA used as reference.

FIG 4

Verification of the integration and excision of the recombinant plasmid pBU4-B3, which carries attP and CDS141 to CDS143. (A) Schematic diagram of pBU4-B3 integration and excision within the transformant. The recombinant plasmid pBU4-B3 contains the 3,963-bp DNA fragment amplified from pTAND672-2, which consists of attP, CDS141, CDS142, and CDS143. As indicated in Fig. 2, the primer pairs Int-a/Int-b and Tet-F/Int-d can be used to detect the attL and attR′ for integration and Int-a/Int-d to detect the attB for excision situation. (B) PCR detection of pBU4-B3 integration and excision within G725Δ0498. M, Trans2k DNA marker; lane 1, attB containing products amplified by Int-a/Int-d (1,020 bp); lane 2, attR′ amplified by Tet-F/Int-d (1,956 bp); lane 3, attL amplified by Int-a/Int-b (751 bp). G725Δ0498 containing pBU4 alone or pBU4-B3Δ143 were used as negative controls. (C) Sequencing verification of the integration and circularization capability. Products containing attL and attR′ were sequenced to verify if intact attB in the chromosome of the host is interrupted by linearized pBU4-B3. Dark and gray arrows indicate the sequences from the recipient chromosome and pBU4-B3, respectively.

FIG 5

Phylogenetic analysis of Int143. The tree was constructed using the neighbor-joining method (p-distance method with a bootstrap of 1,000) and visualized with iTOL (https://itol.embl.de). Red and blue lines in group A indicate 14 predicted integrases/recombinases in the chromosome of the recipient L. sphaericus C3-41 and 23 reference sequences from representative tyrosine recombinase superfamily proteins, respectively. XerA of M. thermautotrophicus Delta H (UniProt accession number O26979.1) displays the highest identity of 44% to Int143, followed by XerD of L. sphaericus C3-41 (GenBank accession number ACA39305.1) (41%) in group A. Int143 was grouped in “clade a” within group B.

FIG 6

Genome comparison of eight B. cereus group plasmids with similar replication, conjugation, and integrase systems to pTAND672-2. The comparison was performed using EasyFig with tBlastX. The shared regions involved in conjugation, putative aimP-aimR arbitrium, replication, and integrase systems are marked with blue, orange, green, and pink boxes, respectively. The mosquitocidal toxins are indicated in red.

FIG 7

Transcriptional analysis of mosquitocidal toxins encoded by BS-pTAND672-2 at different times. Relative RNA expression levels of cry4Aa, cry10Aa, and bin toxins at 12, 24, 48, and 72 h were analyzed by qRT-PCR. The recipient G725Δ0498 was used as the calibrator for the transconjugant.

FIG 8

Electron microscopic observation of crystals produced by strains during sporulation. Crystals of recipient L. sphaericus G725Δ0498 (a), donor B. thuringiensis TAND672 (b), and transconjugant BS-pTAND672-2 (c to d). Red and green arrows indicate toxins specifically produced by B. thuringiensis and L. sphaericus, respectively, while yellow arrows refer to the spore-coat formed by Bacillus isolates. Scale bars are 500 nm.