Isolation of the Bacillus thuringiensis plasmid carrying Bacthuricin F4 coding genes and evidence of its conjugative transfer

Abstract

Introduction: Conjugation is an excellent natural mode of DNA transfer in vivo between bacteria, particularly when these conjugative elements carry technological traits such as bacteriocin encoding genes. In the present work, the bacteriocinogenic plasmid pIBF4 from Bacillus thuringiensis responsible of Bacthuricin F4 synthesis was isolated and characterized. Methodology: To isolate pIBF4, the total plasmid DNA from a non-bacteriocin transposant carrying the mini-Tn10 spectinomycin selective marker was extracted and used to transform Escherichia coli strain Top10. PIBF4 was extracted from the obtained transformant and then subjected to restriction enzyme analysis. Plasmid curing experiments were conducted to test the stability of pIBF4 at a stringent temperature of 42°C. Conjugative behavior of pIBF4 was assessed by mating experiments using the non-bacteriocin transposant mutant as a donor strain and several Bacillus thuringiensis strains as recipients. Results: The pIBF4 plasmid was isolated and had a molecular weight of 19.1 kb. Ninety-five percent of cells retained the pIBF4 plasmid after 200 generations, demonstrating its high stability. PIBF4 was successfully transferred to Bacillus thuringiensis HD1CryB strain with a transfer frequency of 110 -8 transconjugants per donor cell. The study of the recipient host range revealed that pIBF4 is specifically transferable to Bacillus thuringiensis strains with variable transfer frequencies depending on the recipient host strain. Conclusion: Our results show that pIBF4 is a 19.1 kb highly stable plasmid transferable by conjugation to Bacillus thuringiensis strains with deferent transfer frequencies.

Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs

“…tetracycline resistance in plasmid pBC16 (Palva et al., ) and heavy metal resistance genes, e.g. mercury resistance (Belliveau and Trevors, ). Genetic determinants of NRPS (non‐ribosomal peptide synthetases) antagonistic molecules active on other bacteria, fungi, protozoa or other (micro‐)organisms (Zhao et al., ; Kevany et al., ). The genes of bacteriocins such as Thuricin Bn1 (Ugras et al., ), entomocin 110 (Cherif et al., ) or Bacthuricin F4 (Ben Fguira et al., ). The presence of numerous MGE (mobile genetic elements), including IS (insertion sequences) (Léonard et al., ), MICs (mobile insertion cassettes) (Chen et al., ), Tn (transposons) (Mahillon and Lereclus, ; Baum, ; Van der Auwera and Mahillon, ), or group I and group II introns (Tourasse and Kolstø, ).…”

Conjugation across Bacillus cereus and kin: A review

“…Although recent works keep focusing on the importance (Bolotin et al, 2017;Gillis et al, 2018;Fayad et al, 2019Fayad et al, , 2021aZheng et al, 2020), few studies have investigated conjugative transfer per se. Furthermore, reported transfer events often consist in mobilization events without incriminating the implicated conjugative plasmid (Fguira et al, 2014). Therefore, only 20 conjugative plasmids of B. cereus s.l.…”

Evolutionary Genomics of Bacillus thuringiensis

“…Besides, the well known Cry Cyt, Vip, and Sip toxins most of the potential beneficial factors harbored on plasmids have been rarely studied and have not fully elucidated yet. However, bacteriocins, toxin-antitoxin systems, and mobile elements have been identified on transposable plasmids which support the advantage of plasmids in B. thuringiensis (Driss et al, 2011;Fguira et al, 2014;Liu et al, 2008).…”