A New Tool for the Flexible Genetic Manipulation of Geobacillus kaustophilus

Ryotaro Amatsu; Kotaro Mori; Shu Ishikawa; Wilfried J J Meijer; Ken-Ichi Yoshida

doi:10.21769/BioProtoc.4502

A New Tool for the Flexible Genetic Manipulation of Geobacillus kaustophilus

Bio Protoc. 2022 Sep 5;12(17):e4502. doi: 10.21769/BioProtoc.4502.

Authors

Ryotaro Amatsu¹, Kotaro Mori¹, Shu Ishikawa¹, Wilfried J J Meijer², Ken-Ichi Yoshida¹

Affiliations

¹ Department of Science, Technology and Innovation, Kobe University, Kobe, Japan.
² Centro de Biología Molecular 'Severo Ochoa', CSIC-UAM Universidad Autónoma Madrid, Canto Blanco, 28049, Madrid, Spain.

Abstract

Geobacillus kaustophilus , a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of Bacillus subtilis can be transferred via pLS20-mediated conjugation resulting in subsequent integration in the genome of G. kaustophilus. Therefore, we have developed a transformation strategy to design an artificial DNA segment on the chromosome of B. subtilis and introduce it into G. kaustophilus . The artificial DNA segment can be freely designed by taking advantage of the plasticity of the B. subtilis genome and combined with the simplicity of pLS20 conjugation transfer. This transformation strategy would adapt to various Gram-positive bacteria other than G. kaustophilus . Graphical abstract.

Keywords: Bacillus subtilis; Conjugation; Geobacillus kaustophilus; Gram-positive; Homologous recombination; Transformation.