Abstract
Magnetotactic bacteria (MTB) are a diverse group of microorganisms capable of using geomagnetic fields for navigation. This magnetotactic behavior can help microorganisms move toward favorable habitats for optimal growth and reproduction. A comprehensive understanding of the magnetotactic mechanism at molecular levels requires highly efficient genomic editing tools, which remain underdeveloped in MTB. Here, we adapted an engineered CRISPR-Cas9 system for efficient inactivation of genes in a widely used MTB model strain, Magnetospirillum magneticum AMB-1. By combining a nuclease-deficient Cas9 (dCas9) and single-guide RNA (sgRNA), a CRISPR interference system was successfully developed to repress amb0994 expression. Furthermore, we constructed an in-frame deletion mutant of amb0994 by developing a CRISPR-Cas9 system. This mutant produces normal magnetosomes; however, its response to abrupt magnetic field reversals is faster than wild-type strain. This behavioral difference is probably a consequence of altered flagella function, as suggested with our dynamics simulation study by modeling M. magneticum AMB-1 cell as an ellipsoid. These data indicate that, Amb0994 is involved in the cellular response to magnetic torque changes via controlling flagella. In summary, this study, besides contributing to a better understanding of magnetotaxis mechanism, demonstrated the CRISPR-(d)Cas9 system as a useful genetic tool for efficient genome editing in MTB.
Highlights
Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that are capable of sensing and changing their orientation in accordance with geomagnetic fields, a behavior known as “magnetotaxis.” This behavior is thought to facilitate the dwelling of MTB within growth-favoring water columns or sediments with vertical chemical stratification (Blakemore, 1975; Faivre and Schüler, 2008; Lin et al, 2014; Chen et al, 2015)
CRISPR interference (CRISPRi) system can be used to knockdown gene expression in eukaryotes and prokaryotes
To first confirm if deficient Cas9 (dCas9) is expressed in bacterial cells upon induction with IPTG, western blot was performed with dCas9/Cas9 antibodies and a band of 160 kDa was observed, corresponding to the anticipated molecular weight of dCas9 protein (Figure 1A)
Summary
Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that are capable of sensing and changing their orientation in accordance with geomagnetic fields, a behavior known as “magnetotaxis.” This behavior is thought to facilitate the dwelling of MTB within growth-favoring water columns or sediments with vertical chemical stratification (Blakemore, 1975; Faivre and Schüler, 2008; Lin et al, 2014; Chen et al, 2015). This unique capability is facilitated by special. The ability to precisely manipulating MTB chromosome is highly desirable in applications ranging from genetic analysis of functional genomic loci to mechanisms of magnetotaxis
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