Experimental analysis of diverse actin-like proteins from various magnetotactic bacteria by functional expression in Magnetospirillum gryphiswaldense

Abstract

To efficiently navigate within the geomagnetic field, magnetotactic bacteria (MTB) align their magnetosome organelles into chains, which are organized by the actin-like MamK protein. Although MamK is the most highly conserved magnetosome protein common to all MTB, its analysis has been confined to a small subgroup owing to the inaccessibility of most MTB. Our study takes advantage of a genetically tractable host where expression of diverse MamK orthologs together with a resurrected MamK LUCA and uncharacterized actin-like Mad28 proteins from deep-branching MTB resulted in gradual restoration of magnetosome chains in various mutants. Our results further indicate the existence of species-specific MamK interactors and shed light on the evolutionary relationships of one of the key proteins associated with bacterial magnetotaxis.

Keywords: Mad28; MamK; actin-like; cytoskeleton; magnetoskeleton; magnetotactic bacteria.

Fig 1

Maximum-likelihood phylogenetic tree of the actin ATPase protein family showing that Mad28 proteins of magnetotactic Thermodesulfobacteriota and Nitrospirota form a distinct clade compared to other actin-related proteins like MamK, MreB, and FtsA found in magnetotactic bacteria. The tree was drawn to scale, and circles represent statistical support estimated from 500 non-parametric bootstraps. Only significant values above 80 are shown. The sequence database was inspired from that of Monteil and colleagues (4) where accession numbers of crenactin family (Cren) and eukaryotic actin 1 (ACT1) can be found. Here, the MreB family was used to root the tree, since it forms the most ancestral monophyletic group of Actin like proteins based on Ettema and colleagues (10).

Fig 2

(A and B) Schematic representation of magnetosome organization in WT _Mgryph and the magnetoskeleton mutants ∆mamK_Mgryph , ∆mamKY_Mgryph , and ∆mamY_Mgryph . WT, magnetosomes organized in straight chains that run along the geodetic cell axis; ∆mamK_Mgryph , short, fragmented, off-center chains; ∆mamKY_Mgryph , agglomerated magnetosomes or magnetic flux-closed rings; ∆mamY_Mgryph , magnetosome chain at the negative inner cell curvature. Arrows indicate expected reconstitution of magnetosome chain organization in case of successful trans-complementation with mamK from diverse MTB in ∆mamK_Mgryph (A) and in ∆mamKY_Mgryph (B). (C) Degree of complementation as inferred from MC organization in ∆mamK_Mgryph and ∆mamKY_Mgryph . Blue bars, frequency of magnetosome chains positioned at mid-cell in WT _Mgryph , ∆mamK_Mgryph , and ∆mamK_Mgryph that was complemented with the indicated genes. Green bars, frequency of magnetosome chains (>10 particles, resembling ∆mamY_Mgryph phenotype) in ∆mamKY_Mgryph and ∆mamKY_Mgryph that was trans-complemented with the indicated genes. The numbers of analyzed cells (N) correspond to >200. All values are expressed as mean ± standard error of the mean (SEM). Statistical significance is indicated as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and non-significant (ns) when P > 0.05. Statistical significance was assessed using a non-paired t-test between the control strains (ΔmamK_Mgryph in blue and ΔmamKY_Mgryph in green) and the corresponding trans-complemented mutants in the control strains. (D) Relative magnetic response (C_mag ) of complemented ∆mamK_Mgryph and ∆mamKY_Mgryph strains. The C_mag of Mgryph strains that were complemented with the native allele was set at 100%, and the C _mag of the strains with diverse mamKs, mamK _LUCA, mamKJ-like_MV-1, mad28, and parM genes was normalized to the respective value to obtain the relative C _mag as indicator for functional complementation. The values of C_mag were derived from four independent Tn5 insertion mutants.

Fig 3

Three-dimensional structured illumination fluorescence microscopy micrographs (maximum intensity projection, brightfield as inset) of E. coli and Mgryph cells expressing EGFP-MamK/EGFP-Mad28 from diverse MTB and EGFP-ParM _E.coli . Gene expression in E. coli was induced with anhydrotetracycline for 6 hours (50 ng/mL). In ΔmamK_Mgryph , genes were constitutively expressed from P _{mamDC 45}. Scale bars correspond to 1 µm.

Fig 4

(A) Three-dimensional structure model of Mad28 from BW-1 predicted by Alphafold (https://www.rbvi.ucsf.edu/chimerax). The four actin domains, Ia, Ib, IIa, and IIb, are indicated in the structure. (B) SDS-PAGE of the purified N-terminal His-tagged Mad28_BW-1 from E. coli. (C) In vitro polymerization of Mad28_BW-1 in the presence of ATP-γ-S and without ATP (D) visualized by TEM. Scale bar, 100 nm.