Table 1.
Genomic data used in this study.
Table 2.
Primer sets used for PCR amplifications.
Table 3.
A new set of magnetotaxis-related genes outside the M. magneticum AMB-1 magnetosome island.
Figure 1.
Genetic organization of the magnetotaxis islet (MIS) compared to the magnetosome island (MAI) in M. magneticum AMB-1. Each mam gene in the MAI and its respective homologue in the MIS are shown in the same colour. Scale bars: 1 kb. A) The region including mam-like genes is situated between nucleotides (nt) 421600 and 450000 on the bacterial chromosome. For clarity ORFs unrelated to mam genes are not shown. B) Only the mamGFDC and mamAB operons from the MAI (nt 997403 to 1095895) are shown.
Figure 2.
mamK-like transcription in M. magneticum AMB-1 WT and ΔmamK mutant strains.
A) Negative controls (no RT). B) RT-PCR amplification from WT (a–c) and ΔmamK (d–f) RNAs of mamK (b, e) and mamK-like (c, f). mamE was amplified (a, d) as an internal control. Sizes of DNA markers are given on the left on both panels.
Figure 3.
MamK-like belongs to the actin-like protein family.
A) MamK homologue protein sequence alignment (ClustalW) for the Magnetospirillum genus. Accession numbers are Q2W8Q6 (AMB-1) and Q6NE59 (MSR-1) in TrEMBL, and ZP_00054405 (MS-1) in NCBI Refseq. *, conserved residues involved in ATP-binding. B) 3D-structural model of MamK-like (blue) and MamK (cyan) generated by 3D-JIGSAW using the structure of MreB (red) from Thermotoga maritime (PDB entry: 1JCF) as a template. The ATP molecule is shown in violet.
Figure 4.
SDS-PAGE and Western blot detection of purified recombinant MamK-like and MamK.
A) SDS-PAGE gel (10% acrylamide) with 2 µg of protein per lane stained with Coomassie blue. B) Western blot with 30 ng of protein per lane probed with anti-histidine tag antibody. C) Western blot with 30 ng of protein per lane probed with anti-MamK antibody. Molecular weights (MW) of protein standards are given on the left and apply to all panels. Expected MWs of MamK-like and MamK are 41 and 42 kDa respectively.
Figure 5.
In vitro polymerization of MamK and MamK-like visualized by TEM.
Sizes of structures are indicated with arrows. Structures narrower than 10 nm are termed “filaments” and larger structures are termed “bundles”. A–B) MamK polymers. C–D) MamK-like polymers. Scale bars: 100 nm in A, B and D; 300 nm in C.
Figure 6.
In vivo polymerization kinetics of MamK-like-Venus and MamK-Venus filaments in E. coli measured by fluorescence imaging.
A) Polymerization kinetics. Protein production was induced by adding 0.1% arabinose during the exponential growth phase. Left panel, fluorescence emission at 528 nm (515 nm excitation); middle panel, Nomarski contrast; right panel, left and middle images overlaid. Scale bars: 5 µm. Time after induction is given on the left. B) Comparison of filament morphology of MamK-like-Venus and MamK-Venus after 3 h of induction with 0.2% arabinose. Left panel, fluorescence emission at 528 nm (515 nm excitation) after deconvolution; middle panel, Nomarski contrast; right panel, left and middle images overlaid. Scale bars: 3 µm.
Figure 7.
Detection of MamK and MamK-like filaments in fixed cells of M. magneticum AMB-1 WT and ΔmamK strains.
The primary antibody was the anti-MamK antibody and the secondary antibody was coupled to FITC. Confocal microscope data acquisition parameters: 3D stacks of 2048 x 2048 pixel images, 0.16 µm steps, 4x frames average, 2x line accumulation. A) FITC fluorescence emission at λ = 518 nm (excitation at λ = 488 nm), 2.4x microscope zoom. Left panel, ΔmamK mutant cells; right panel, WT cells. Scale bar: 10 µm. B) Cell membranes were stained with FM® 4-64 FX. Upper panels, ΔmamK mutant cells; lower panels, WT cells. Left column, FITC fluorescence emission at λ = 518 nm (excitation at λ = 488 nm); middle column, FM® 4-64 FX fluorescence emission at λ = 744 nm (excitation at λ = 633 nm); right column, left and middle column images overlaid; 4.74x microscope zoom for all panels. Scale bar: 1 µm. C) Visualization of magnetosome alignment with TEM. Left panel, ΔmamK cells (unstained); right panel, WT cells (1% uranyl acetate stained). Scale bar: 300 nm.
Figure 8.
RSCU-based trees generated with the Fitch-Margoliash distance-based optimization method. The best trees were computed by the algorithm from 100 randomized input order distance matrixes. A) mamD gene family. B) mamE gene family. C) mamK gene families. MamE-II (for MS-1 and AMB-1) are MamE homologues encoded in the MAI. Sub-trees comprising mam-like genes from the AMB-1 magnetotaxis islet are shaded grey. Boxed gene names are the mam genes belonging to the AMB-1 magnetosome island. Accession numbers for the encoded proteins are listed in Table S1.
Figure 9.
Kinship of MIS mam-like genes with MAI mam genes of M. gryphiswaldense MSR-1.
A) Partial sequence alignment of MamE proteins from the Magnetospirillum genus. B) Protein sequence alignment between MamQ-like encoded in the MIS of AMB-1, the newly identified MamQ-like from MSR-1 (MGR_0326) and MamQ encoded in the MAI of AMB-1.