25 nm

Images: D. Schuler (inset), T. Beveridge (background)

Biomineralization of Nanocrystalline Magnetite In BacteriaArash Komeili

Department of Plant and Microbial BiologyUC Berkeley

Magnetotactic Bacteria

• Morphologically and phylogenetically diverse• Orient in magnetic fields using magnetosomes• Use geomagnetic fields for direction-sensing

Magneto-aerotaxis

• Frankel, Bazylinksi, Johnson and Taylor. Biophys Journal 1997• Smith et al. Biophys Journal 2007

D. Schuler

Why Study Magnetotactic Bacteria?

-Geobiology “magnetofossils” as biomarkers

-Applications Biomedicine, biotechnology, bioremediation

-Biomineralization Genetic control of crystal properties

• Lipid Compartment

• Unique set of proteins

• Organized into chains with a cytoskeleton

25 nm

Magnetite Crystals are Formed Within Lipid Compartments

D. Schuler

Dr. Grant Jensen

Dr. Zhuo Li

Division of Biology, California Institute of Technology

Electron cryo-tomography of Magnetospirillum magneticum AMB-1

Komeili, Li, Newman, Jensen. Science 2006

Movie by Zhuo Li and Grant Jensen

Model for Magnetosome Formation

Model for Magnetosome Formation

What are the genes and proteins that control these various functions?

Approaches for Identifying Magnetosome Biogenesis Factors

Induction of Magnetite Synthesis

+Fe -Fe

Genetic Screen Using a Magnetic Plate System

0 20 seconds 5 minutes

The Mutants

Grunberg et al. AEM (2001)

mnm2 mnm3

WT mnm1 mnm2 mnm3

Magnetosome Gene Island Contains the Majority of Magnetosome Genes

Fukuda et al. FEBS Letters, Feb. 2006

Stats: 98 kb, 106 genes(or more!), 2% of the genome

Aims:

• Identify genes involved in various steps of magnetosome formation

• Define a minimum set of genes sufficient for magnetosome formation

• Investigate the evolution and diversity of magnetosome formation

Work by: Dr. Dorothee Murat

Genetic Dissection of the Magnetosome Island

Anti-MamK Anti-MamC

WT MaI 1 7 5 6WT MaI 1 7 5 6 WT MaI 1 7 5 6

Anti-MamA

• Deletion of all 18 genes eliminates all traces of magnetosomes.• Next step: generate individual deletions of these 18 genes.• Each gene has a distinct role:

• Membrane Formation• Chain Formation• Biomineralization

mamAB Gene Cluster is a Central Regulator of Magnetosome Formation

MamI, MamL, MamQ and MamB are essential for membrane biogenesis

DmamL strainH. Vali

0.2 µm

MamK Aligns Magnetosomes Into Chains

Biomineralization Mutants

mamV mutant

mamS mutant

What are the magnetic signatures of these mutants? ALS collaboration with Marco Liberati.

Model for magnetosome formation

MamK, MamJ

MamC, MamD, Mms6, MamA

Komeili et al. PNAS 2004Komeili et al. Science 2006Scheffel et al. Nature 2006Arakaki et al. JBC 2003

Model for magnetosome formation

R2, Mam I, MamL, MamQ, MamB, MamE (?)

MamK, MamJ

MamC, MamD, Mms6, MamE, MamO MamMN, MamSTU, MamA, MamPR2, R3

?

Future Directions

• Cell biological characterization of magnetosome formation

• Identification of a minimum set of genes sufficient for magnetite formation

• Evolution and diversity of magnetotactic bacteria

Branches which contain magnetotactic bacteria (MTB)

MTB which grow in culture and are sequenced

One strain of magnetotactic bacteria outside the α-Proteobacteriahas been cultured and sequenced

α-Proteobacteriaδ-Proteobacteria

NitrospiraMagnetococcus MC-1

MMP

Magnetobacterium bavaricum

γ-Proteobacteria

Burkholderia cepacia

Shewanella alga

β-Proteobacteria

Desulfosarcina variabilis

Geobacter metallireducens

Desulfovibrio sp. BG6Desulfovibrio RS-1

Desulfovibrio desulfuricans

Nitrospira moscoviensis

Leptospirillum ferrooxidans

Agrobacterium tumefaciens

Oceanospirillum pusillumMagnetic vibrio MV-1

MSM-3 and MSM-4

Magnetospirillum AMB-1

Magnetospirillum MS-1

Phaeospirillum moüsckianumMagnetospirillum MRS-1

Rhodospirillum rubrum

CS92

CS103TB12

CS308TB24

MP17

Itaipu Imacpal19

CS81

macpal9

macpal1

Cyanobacteria

Planctomycetes

Thermotoga maritima

Aquifex pyrophilus Archaea10%

Itaipu II

tree from Amman et al. in Biomineralization 2004

Desulfovibrio sp. RS-1

0.1 μm

RS-1

MS-1

Taoka et al. 2006

Arakaki et al. 2003

AMB-1

Grunberg et al. 2004

MSR-1

MC-1

Meldrum et al. 1993

Desulfovibrio magneticus sp. RS-1 forms crystals with a different morphology

What cellular structures are involved in biomineralization of magnetite crystals in RS-1?

Komeili et al. 2006

Magnetite crystals from cultured α-Proteobacterial magnetotactic bacteria

Hours after iron addition

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Fe-P granulesMagnetite crystals

RS-1 forms round granules then magnetite crystalsafter addition of iron following iron starvation

At left are TEM images of whole RS-1 cells at different times after the addition of iron. At three hours, we begin to see the formation of round granules – which have not been seen previously in magnetotactic bacteria. By 22 hours, we begin to see magnetite crystals form, and by 50 hours the granules have disappeared and we see only magnetite crystals. At right, we have plotted the average number of granules and magnetite crystals per cell over time.

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The granules are non-crystallineand contain iron and phosphate

High-resolution TEM:

magnetite crystals granules

Elemental analysis (Energy-dispersive X-ray Spectoscopy) showed that the granules contain primarily iron and phosphorus.

Cryo electron microscopy shows membranes around iron-phosphate granules but not magnetite crystals

100 nm 100 nm

Iron-phosphate granules Magnetite crystals

Arrow highlights membrane.

Electron tomography of RS-1 thin section also showsno membranes around magnetite crystals

Conclusions:

• Magnetite crystals in RS-1 do not seem to form within membrane-bounded compartments. Thissuggests that RS-1 forms crystals using a distinct mechanism, perhaps a protein template.

• RS-1 forms non-crystalline iron-phosphate granules within membrane-bounded compartments. These compartments constitute a novel bacterial organelle.

Caltech:Dianne NewmanMel SimonGrant JensenZhuo Li Cody Nash

McGill university:H. Vali

Funding:

• David and Lucille Packard Foundation

• Hellman Family Fund

• NIH

Komeili Lab:

Olga DraperMeg ByrneDorothee MuratAnna QuinlanShannon GreeneSepehr KeyhaniJoyce Cueto