A cumate-inducible promoter system for tuned gene expression and magnetosome overproduction in magnetotactic bacteria

Abstract

Magnetosomes are membrane-bound iron-rich organelles synthesized by magnetotactic bacteria as sensors for navigation in the Earth’s magnetic field. In the well-studied model Magnetospirillum gryphiswaldense, their multistep biosynthesis is tightly controlled by > 30 specific genes clustered within a genomic magnetosome island. The lack of versatile genetic tools for strong, tunable, and orthogonal transcriptional control has impeded the analysis, engineering, and transplantation of this complex biosynthetic pathway. In addition, recent studies revealed an intricate transcriptional architecture of magnetosome gene clusters, but its significance has still remained unclear. Here, by using a bioluminescence reporter assay, we identify several novel constitutive promoters and establish the cumate-inducible repressor/promoter system CymR/Pcym as a robust and tunable regulatory module for rapid, dose-dependent transcriptional control in M. gryphiswaldense. We demonstrate its successful application for genetic tuning of magnetosome biosynthesis and inducible overproduction by controlling expression of a single essential magnetosome gene in a duplicated hybrid magnetosome gene cluster background. Additionally, we show its versatility by creating a hybrid promoter within the essential long mamAB operon, which experimentally confirms the significance of the intragenic P(mamH) promoter for the generation of long polycistronic transcripts. In summary, our data underlines the importance of balanced magnetosome gene expression. We also provide a versatile framework for dynamic transcriptional control and genetic engineering strategies in M. gryphiswaldense and other bacteria.

Graphical abstract

Keypoints • Several novel constitutive promoters expand the genetic toolbox of M. gryphiswaldense. • CymR/Pcym allows strong, controlled and tunable magnetosome gene expression. • A hybrid inducible magnetosome overproducer shows increased stability.