Development of a Gene Replacement Method for the Filamentous Bacterium Leptothrix cholodnii SP-6

Tatsuki Kunoh; Erika Ono; Tatsuya Yamamoto; Ichiro Suzuki; Minoru Takeda; Nobuhiko Nomura

doi:10.21769/BioProtoc.4652

Development of a Gene Replacement Method for the Filamentous Bacterium Leptothrix cholodnii SP-6

Bio Protoc. 2023 Apr 20;13(8):e4652. doi: 10.21769/BioProtoc.4652.

Authors

Tatsuki Kunoh¹, Erika Ono², Tatsuya Yamamoto¹, Ichiro Suzuki³, Minoru Takeda³, Nobuhiko Nomura^{1

4}

Affiliations

¹ Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 Japan.
² Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 Japan.
³ Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya, Yokohama 240-8501 Japan.
⁴ Microbiology Research Center for Sustainability (MiCS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 Japan.

Abstract

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteriaLeptothrix cholodniiSP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation withEscherichia coliS17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes. Graphical overview.

Keywords: Calcium depletion; Conjugation; Escherichia coliS17-1; Gene transfer; Leptothrix cholodniiSP-6.