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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 Fe2+
compounds, which form extracellular finegrained 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 Fe2+ detected (evidence of iron
reduction)
Day 3 to 5:
intracellular granules observed (always after
appearance of Fe2+)
Day 14:
more than 90% of population contained
intracellular granules
Change in Fe2+ 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
• 30-50 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
Conditions
Ferrihydrite
Iron
Reduction
Yes
Presence of Growth
Granules
Yes
Yes
(anaerobic)
Goethite
No
No
No
No
No
No
No
No
Yes
No
No
Yes
(anaerobic)
Hematite
(anaerobic)
Fumarate
(anaerobic)
Aerobic
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 magnetsensitive 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
Fe2+
• 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 magnetsensitive
• 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