Intracellular Iron Minerals in a Dissimilatory Iron-Reducing Bacterium Susan Glasauer,* Sean Langley, Terry J. Beveridge

“What do we have here?” Shewanella spp. are Gram-negative facultative anaerobic bacteria It can use iron (III) oxides as terminal electron acceptor (as opposed to oxygen) Fe (III) compounds are reduced to Fe 2+ compounds, which form extracellular fine- grained minerals Point of Interest! Intracellular granules are observed in Shewanella putrefaciens following iron reduction This could represent an unexplored pathway for the cycling of iron by bacteria

Exp.1: “Show me the Granules!” Investigation of structure and content Shewanella putrefaciens CN32 is grown anaerobically on defined medium containing two-line ferrihydrite as an iron source Fe concentrations representative of natural levels

S. putrefaciens CN32 in Action Immediate adsorption of ferrihydrite to cells Day 1 to 4: significant Fe 2+ detected (evidence of iron reduction) Day 3 to 5: intracellular granules observed (always after appearance of Fe 2+ ) Day 14: more than 90% of population contained intracellular granules

Change in Fe 2+ concentration (diamonds) and cell numbers (circles) over time for anaerobic liquid cultures of S. putrefaciens CN32 during growth in a defined medium with two-line ferrihydrite.

Intracellular Granule Observations nm Concentrated in cytoplasm at one or both cell poles Formed near plasma membrane, pushed into cytoplasm Regular polyhedral shape Homogenously distributed Number of granules per cell increased with time to about 60 per cell Cells with granules appeared healthier

Intracellular fine-grained granules concentrated at poles; enclosed in membranes

Intracellular Granule Observations TEM Membrane bound (membrane may assist the cell in manufacturing of intracellular mineral) EDS Rich in iron and oxygen with no other metals or counterions SAED Diffraction pattern similar to ferrihydrite, however rings observed were broader and more diffused 10% of granule clusters showed patterns distinct from ferrihydrite. Mineral could not be identified exactly; similarity to magnetite and maghemite noted.

Energy-dispersive x-ray spectrum from an intracellular granule formed by S. putrefaciens CN32 (a), and granule-free portion of the cell (b), elucidating composition of formed granules.

Extracellular Observations At 7 days (SAED): Randomly oriented ferrihydrite only 7 to 46 days (XRD, SAED): Green rust, magnetite, vivianite, goethite, poorly crystalline Fe phases Abiotic control shows presence of ferrihydrite only This suggests that Shewanella putrefaciens CN32 regulates Fe geochemistry.

Extracellular two-line ferrihydrite adsorbed to cell

Exp.2: Variable Electron Acceptors ConditionsIron Reduction Presence of Granules Growth Ferrihydrite (anaerobic) Yes Goethite (anaerobic) No Hematite (anaerobic) No Fumarate (anaerobic) No Yes AerobicNo Yes

Exp.3: Variations in Ferrihydrite Concentrations Affected iron reduction rate Affected appearance of granules Did not affect intracellular granule formation Exp.4: Variations in Inoculum Density Affects rate of iron reduction Affects amount of intracellular minerals formed per cell Suggests that actual rate of iron reduction per cell remains constant

Intracellular Iron Granules: Magnetism? Organisms within the Proteobacteria showed magnet- sensitive properties in intracellular structures Shewanella is part of the Proteobacteria group, its intracellular granules may exhibit magnetic properties Tests proved to be inconclusive: –Extracellular magnetite prevented determination of possible magnetic properties of inclusions –Granules in Shewanella putrefaciens CN32 differed in appearance, composition and location in cell compared to that of the magnetic granules of other organisms

Function of Intracellular Granules: …Unknown… Easy source of iron? Size and morphology inconsistent with that function Affects behavior? Similar in content with granules found in abdominal cells of honeybees (Kuterbach suggests that they may play a role in orientation)

Conclusions Granules containing iron and oxygen formed intracellularly after ferrihydrite was reduced to Fe 2+ There is a membrane surrounding each intracellular granule The cells are probably responsible for regulation of Fe geochemistry Only the ferrihydrite form of the ferric (hydr)oxides tested can function as the electron acceptor for S. putrefaciens and still allow cell growth and granule formation

Criticisms Significance of extracellular iron compounds formed not explained Paper mentioned membranes of granules could assist in granule formation – no obvious justification for this speculation Not specified whether ferrihydrite was present when S. putrefaciens was grown under aerobic conditions in exp. 2 Natural growth conditions of bacteria unspecified Authors assume in depth knowledge of diffraction methods Why were they grown first aerobically, then transferred to anaerobic conditions for exp. 1? How does cytoplasm density correlate to the healthiness of the cell?

To be continued… Determine process by which intracellular granules formed Determine functional significance of intracellular granules Determine whether intracellular granules are magnet- sensitive Identify a mineral present in a small fraction of the intracellular granules Determine whether the granule membrane helps the cell to manufacture the new intracellular mineral Test other ferric oxide compounds for ability to act as electron acceptors and ability to form intracellular granules

Presented by: Yvonne Chan Arina Kondrakhina