Stable and efficient delivery of DNA to Bacillus subtilis (natto) using pLS20 conjugational transfer plasmids

Mitsuhiro Itaya, Mayumi Nagasaku, Tomoe Shimada, Naoto Ohtani, Yuh Shiwa, Hirofumi Yoshikawa, Shinya Kaneko, Masaru Tomita, Mitsuru Sato

Research output: Contribution to journalArticle

Abstract

Bacillus subtilis (natto) is generally regarded as a safe bacterium and used as a host for the production of several materials. However, genetic engineering of B. subtilis (natto) is not well established because of poor DNA delivery methods and the lack of a standard strain for the aim. Here, we developed a genetic delivery tool in B. subtilis (natto) using the pLS20 conjugational plasmid (65 kbp). Transmission of pLS20 from B. subtilis 168 to wild-type B. subtilis (natto) did not occur via established mating protocols. We isolated B. subtilis (natto) mutants showing dramatically increased recipient activity. Whole-genome sequence analyses revealed three common alterations: mutations in the restriction endonuclease gene and in the methyl-accepting chemotaxis protein gene, and a 43-kbp deletion at the genome replication termination locus. A representative strain named NEST116 was generated as the first B. subtilis (natto) strain suitable for exploring pLS20-based genetic engineering.

Original languageEnglish
JournalFEMS microbiology letters
Volume366
Issue number4
DOIs
Publication statusPublished - 2019 Feb 1

Fingerprint

Bacillus subtilis
Plasmids
DNA
Genetic Engineering
Genome
DNA Restriction Enzymes
Genes
Sequence Analysis
Bacteria
Mutation

Keywords

  • B. subtilis
  • B. subtilis (natto)
  • antibiotic selection marker
  • conjugational transfer
  • green fluorescence
  • restriction modification

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Genetics

Cite this

Stable and efficient delivery of DNA to Bacillus subtilis (natto) using pLS20 conjugational transfer plasmids. / Itaya, Mitsuhiro; Nagasaku, Mayumi; Shimada, Tomoe; Ohtani, Naoto; Shiwa, Yuh; Yoshikawa, Hirofumi; Kaneko, Shinya; Tomita, Masaru; Sato, Mitsuru.

In: FEMS microbiology letters, Vol. 366, No. 4, 01.02.2019.

Research output: Contribution to journalArticle

Itaya, Mitsuhiro ; Nagasaku, Mayumi ; Shimada, Tomoe ; Ohtani, Naoto ; Shiwa, Yuh ; Yoshikawa, Hirofumi ; Kaneko, Shinya ; Tomita, Masaru ; Sato, Mitsuru. / Stable and efficient delivery of DNA to Bacillus subtilis (natto) using pLS20 conjugational transfer plasmids. In: FEMS microbiology letters. 2019 ; Vol. 366, No. 4.
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