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A second actin-like MamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island.

Rioux JB, Philippe N, Pereira S, Pignol D, Wu LF, Ginet N - PLoS ONE (2010)

Bottom Line: In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and DeltamamK mutant cells and that the actin-like filamentous structures observed in the DeltamamK strain are probably MamK-like polymers.Thus MamK-like is a new member of the prokaryotic actin-like family.This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Bioénergétique Cellulaire - Institut de Biologie Environnementale et Biotechnologie, Commissariat à l'Energie Atomique, Saint-Paul-lez-Durance, France.

ABSTRACT
Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and DeltamamK mutant cells and that the actin-like filamentous structures observed in the DeltamamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.

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Related in: MedlinePlus

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.
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pone-0009151-g004: 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.

Mentions: Recombinant histidine-tagged MamK and MamK-like proteins were produced in E. coli and purified by metal affinity chromatography (Fig. 4A). Both purified proteins were detected by Western blot with an antibody raised against MamK (Fig. 4C), though detection of MamK-like was less sensitive. Size-exclusion experiments (data not shown) using different buffers revealed that addition of ATP is required to obtain stable monomers and to reduce polymerization and/or precipitation of both proteins during purification. To investigate this and test the ability of MamK and MamK-like to spontaneously form filaments, purified proteins were incubated in different buffers and analyzed after different time lapses by negative stain TEM. As previously shown for MamK [24], MamK-like is able to spontaneously polymerize into long straight filaments in the absence of ATP, a process triggered by the addition of KCl and MgCl2 (Fig. 5). MamK and MamK-like form large structures, we termed “bundles”, approx. 60 nm in width and reaching more than 1 µm in length. Smaller assemblies ranging from 20 to 35 nm in width can also be observed (Fig. 5). Small bundles are made up of individual filaments whose size (diameters from 6 to 8 nm) and striated appearance are consistent with them being elemental helical filaments described previously [24]. Small MamK and MamK-like bundles are organized differently. MamK bundles are composed of twisted filaments, whereas MamK-like filaments are assembled in a more regular, linear bundle. On a larger scale, well-developed bundles of MamK-like are also generally straighter and more regularly arranged than MamK bundles (Fig. 5A,C). Another major difference between the two proteins is in the in vitro polymerization kinetics since MamK-like bundles are only observed after 30 minutes of incubation whereas MamK polymerizes in less than 15 min.


A second actin-like MamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island.

Rioux JB, Philippe N, Pereira S, Pignol D, Wu LF, Ginet N - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2818848&req=5

pone-0009151-g004: 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.
Mentions: Recombinant histidine-tagged MamK and MamK-like proteins were produced in E. coli and purified by metal affinity chromatography (Fig. 4A). Both purified proteins were detected by Western blot with an antibody raised against MamK (Fig. 4C), though detection of MamK-like was less sensitive. Size-exclusion experiments (data not shown) using different buffers revealed that addition of ATP is required to obtain stable monomers and to reduce polymerization and/or precipitation of both proteins during purification. To investigate this and test the ability of MamK and MamK-like to spontaneously form filaments, purified proteins were incubated in different buffers and analyzed after different time lapses by negative stain TEM. As previously shown for MamK [24], MamK-like is able to spontaneously polymerize into long straight filaments in the absence of ATP, a process triggered by the addition of KCl and MgCl2 (Fig. 5). MamK and MamK-like form large structures, we termed “bundles”, approx. 60 nm in width and reaching more than 1 µm in length. Smaller assemblies ranging from 20 to 35 nm in width can also be observed (Fig. 5). Small bundles are made up of individual filaments whose size (diameters from 6 to 8 nm) and striated appearance are consistent with them being elemental helical filaments described previously [24]. Small MamK and MamK-like bundles are organized differently. MamK bundles are composed of twisted filaments, whereas MamK-like filaments are assembled in a more regular, linear bundle. On a larger scale, well-developed bundles of MamK-like are also generally straighter and more regularly arranged than MamK bundles (Fig. 5A,C). Another major difference between the two proteins is in the in vitro polymerization kinetics since MamK-like bundles are only observed after 30 minutes of incubation whereas MamK polymerizes in less than 15 min.

Bottom Line: In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and DeltamamK mutant cells and that the actin-like filamentous structures observed in the DeltamamK strain are probably MamK-like polymers.Thus MamK-like is a new member of the prokaryotic actin-like family.This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Bioénergétique Cellulaire - Institut de Biologie Environnementale et Biotechnologie, Commissariat à l'Energie Atomique, Saint-Paul-lez-Durance, France.

ABSTRACT
Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and DeltamamK mutant cells and that the actin-like filamentous structures observed in the DeltamamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.

Show MeSH
Related in: MedlinePlus