Biological Containment of Genetically Modified Bacillus subtilis

Siamand Hosseini, Aleksandrs Curilovs, Simon Cutting

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Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores coupled with their desiccation resistance makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of spore GMOs by utilizing the concept of a ‘thymine-less death’, a phenomenon first reported six decades ago. Using Bacillus subtilis we have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB, using a two-step process. Insertion is made first at thyA followed by thyB where resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine) and in their absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system we have successfully created a number of spore vaccines as well as spores displaying active enzymes.
Original languageEnglish
Article numbere02334-17
Pages (from-to)1-15
Number of pages15
JournalApplied and environmental microbiology
Issue number3
Early online date17 Jan 2018
Publication statusPublished - 2018


  • Bacillus subtilis
  • spores
  • GMOs
  • containment

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