The acidic repetitive domain of the Magnetospirillum gryphiswaldense MamJ protein displays hypervariability but is not required for magnetosome chain assembly

Abstract

Magnetotactic bacteria navigate along the earth's magnetic field using chains of magnetosomes, which are intracellular organelles comprising membrane-enclosed magnetite crystals. The assembly of highly ordered magnetosome chains is under genetic control and involves several specific proteins. Based on genetic and cryo-electron tomography studies, a model was recently proposed in which the acidic MamJ magnetosome protein attaches magnetosome vesicles to the actin-like cytoskeletal filament formed by MamK, thereby preventing magnetosome chains from collapsing. However, the exact functions as well as the mode of interaction between MamK and MamJ are unknown. Here, we demonstrate that several functional MamJ variants from Magnetospirillum gryphiswaldense and other magnetotactic bacteria share an acidic and repetitive central domain, which displays an unusual intra- and interspecies sequence polymorphism, probably caused by homologous recombination between identical copies of Glu- and Pro-rich repeats. Surprisingly, mamJ mutant alleles in which the central domain was deleted retained their potential to restore chain formation in a DeltamamJ mutant, suggesting that the acidic domain is not essential for MamJ's function. Results of two-hybrid experiments indicate that MamJ physically interacts with MamK, and two distinct sequence regions within MamJ were shown to be involved in binding to MamK. Mutant variants of MamJ lacking either of the binding domains were unable to functionally complement the DeltamamJ mutant. In addition, two-hybrid experiments suggest both MamK-binding domains of MamJ confer oligomerization of MamJ. In summary, our data reveal domains required for the functions of the MamJ protein in chain assembly and maintenance and provide the first experimental indications for a direct interaction between MamJ and the cytoskeletal filament protein MamK.

FIG. 1.

C_mag values (average magnetic response) of wild-type (WT), ΔmamJ, and ΔmamJ cells trans-complemented with the full-length wild-type allele (pAS50). Gray bars, motile cells; black bars, formaldehyde-killed cells. Cultures were grown in triplicate and diluted to an optical density at 565 nm of 0.1 prior to C_mag measurements.

FIG. 2.

(A) TEM micrographs of wild-type (I), ΔmamJ (II to VII), and ΔmamJ cells trans-complemented with pAS50 (VIII). Scale bars, 500 nm. (B) Distribution of magnetosome crystal numbers in stationary cultures of wild-type (I) and ΔmamJ (II) cells. Numbers were determined by counting 309 cells by TEM. Insets illustrate the magnetosome distribution to daughter cells during cell division in wild-type and ΔmamJ cells.

FIG. 3.

Domain organization, sequence characteristics (A), and similarity tree (B) of MamJ protein sequences from M. gryphiswaldense MSR-1, M. magneticum AMB-1, M. magnetotacticum MS-1 and Magnetospirillum sp. strains CF-2 and CF-3. MW, molecular weight (in thousands).

FIG. 4.

Ability of various MamJ mutant proteins to restore magnetosome chain formation in ΔmamJ. (A) Overview of constructs used for trans-complementation. Results of chain formation analysis by TEM are indicated by “+” or “−.” Dashed lines indicate deleted sequence fragments. (B) C_mag values (average magnetic response) of formaldehyde-killed ΔmamJ cultures trans-complemented with different MamJ mutant constructs. The dashed line represents a threshold (C_mag = 0.3) that discriminates between functional and nonfunctional MamJ mutant constructs. (C) Immunodetection of wild-type and mutant MamJ proteins. Proteins were expressed in trans in the ΔmamJ mutant and immunodetected by an anti-MamJ peptide antibody in crude extracts resolved by SDS-PAGE. Mutant protein JΔ81-256 lacks the epitope recognized by the antibody. Predicted molecular masses (MW) and isoelectric points are indicated below.

FIG. 5.

Intracellular localization of a MamK-EGFP fusion in ΔmamJ and in E. coli DH5α cells. Fluorescence micrographs of ΔmamJ cells and of DH5α cells stained with the membrane dye FM4-64 (red) and expressing a MamK-EGFP (green) fusion.

FIG. 6.

Two-hybrid analysis of the interaction between MamJ and MamK and MamJ and MamJ. (A) Growth of reporter E. coli spotted onto selective and nonselective screening medium after cotransformation with different bait (derivative of pBT) and prey (derivative of pTRG) expression vectors. Colony growth on nonselective screening medium verifies that cotransformation was successful, whereas on selective screening medium, colonies can grow only in the case of an interaction between bait and prey fusion proteins. (B) Overview of analyzed MamJ sequence regions (prey) for two-hybrid interaction with MamK, MamJ, and the N-terminal part of MamJ (bait). Growth on both selective and nonselective screening medium is shown by +; − indicates growth on nonselective screening medium only. n.d., not determined. (C) Immunodetection of prey fusion proteins by an anti-MamJ peptide antibody in protein crude extracts of the BacterioMatch reporter E. coli resolved by SDS-PAGE. RNAP fusions with J295-334, J330-368, and J361-399 lack the epitope recognized by the antibody.