1. 25 nm Images: D. Schuler (inset), T. Beveridge (background) Biomineralization of Nanocrystalline Magnetite In Bacteria Arash Komeili Department of Plant and Microbial Biology UC Berkeley

2. Magnetotactic Bacteria Morphologically and phylogenetically diverse Orient in magnetic fields using magnetosomes Use geomagnetic fields for direction-sensing

4. Magneto-aerotaxis Frankel, Bazylinksi, Johnson and Taylor. Biophys Journal 1997 Smith et al. Biophys Journal 2007

5. D. Schuler Why Study Magnetotactic Bacteria? -Geobiology “magnetofossils” as biomarkers -Applications Biomedicine, biotechnology, bioremediation -Biomineralization Genetic control of crystal properties

6. Lipid Compartment Unique set of proteins Organized into chains with a cytoskeleton 25 nm Magnetite Crystals are Formed Within Lipid Compartments D. Schuler

7. 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

9. Movie by Zhuo Li and Grant Jensen

12. Model for Magnetosome Formation

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

14. Approaches for Identifying Magnetosome Biogenesis Factors

15. Induction of Magnetite Synthesis +Fe -Fe

17. Genetic Screen Using a Magnetic Plate System 020 seconds5 minutes

18. The Mutants Grunberg et al. AEM (2001) mnm2mnm3 WTmnm1mnm2mnm3

19. 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

20. 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

21. Anti-MamKAnti-MamC 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

22. MamI, MamL, MamQ and MamB are essential for membrane biogenesis  mamL strain H. Vali 0.2 µm

23. MamK Aligns Magnetosomes Into Chains

24. Biomineralization Mutants mamV mutant mamS mutant What are the magnetic signatures of these mutants? ALS collaboration with Marco Liberati.

25. Model for magnetosome formation MamK, MamJ MamC, MamD, Mms6, MamA Komeili et al. PNAS 2004 Komeili et al. Science 2006 Scheffel et al. Nature 2006 Arakaki et al. JBC 2003

26. Model for magnetosome formation R2, Mam I, MamL, MamQ, MamB, MamE (?) MamK, MamJ MamC, MamD, Mms6, MamE, MamO MamMN, MamSTU, MamA, MamP R2, R3 ?

27. 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

28. Branches which contain magnetotactic bacteria (MTB) MTB which grow in culture and are sequenced One strain of magnetotactic bacteria outside the α-Proteobacteria has been cultured and sequenced α-Proteobacteria δ-Proteobacteria Nitrospira Magnetococcus MC-1 MMP Magnetobacterium bavaricum γ-Proteobacteria Burkholderia cepacia Shewanella alga β-Proteobacteria Desulfosarcina variabilis Geobacter metallireducens Desulfovibrio sp. BG6 Desulfovibrio RS-1 Desulfovibrio desulfuricans Nitrospira moscoviensis Leptospirillum ferrooxidans Agrobacterium tumefaciens Oceanospirillum pusillum Magnetic vibrio MV-1 MSM-3 and MSM-4 Magnetospirillum AMB-1 Magnetospirillum MS-1 Phaeospirillum moüsckianum Magnetospirillum MRS-1 Rhodospirillum rubrum CS92 CS103 TB12 CS308 TB24 MP17 Itaipu I macpal19 CS81 macpal9 macpal1 Cyanobacteria Planctomycetes Thermotoga maritima Aquifex pyrophilus Archaea 10% Itaipu II tree from Amman et al. in Biomineralization 2004 Desulfovibrio sp. RS-1

29. 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

30. Hours after iron addition RS-1 forms round granules then magnetite crystals after 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. Number of electron-dense granules per cell

31. The granules are non-crystalline and 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.

32. Cryo electron microscopy shows membranes around iron-phosphate granules but not magnetite crystals 100 nm Iron-phosphate granules Magnetite crystals Arrow highlights membrane.

33. Electron tomography of RS-1 thin section also shows no membranes around magnetite crystals Conclusions: Magnetite crystals in RS-1 do not seem to form within membrane-bounded compartments. This suggests 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.

34. Caltech: Dianne Newman Mel Simon Grant Jensen Zhuo Li Cody Nash McGill university: H. Vali Funding: David and Lucille Packard Foundation Hellman Family Fund NIH Komeili Lab: Olga Draper Meg Byrne Dorothee Murat Anna Quinlan Shannon Greene Sepehr Keyhani Joyce Cueto