Microbiology Journal Club
September 29, 2008
Casey Theriot
What is polyphosphate and why is it
important?
A chain of many phosphates,
joined by a high-energy
phosphoanhydride bond.
Functions of Poly P in the cell
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ATP substitute and energy source
Reservoir for Pi (inorganic phosphate)
Chelator of metal ions
Buffer against alkali
Channel for DNA entry
Regulator for stress and survival
Regulator of development
Cell capsule for select bacteria
Important terms and reactions
•  PPK: polyphosphate kinase, catalyzes the
reversible conversion of terminal
phosphate ATP into polyP
nATP
poly Pn + nADP
•  PPX: exopolyphosphatase, hydrolyzes the
terminal residues of polyP to liberate Pi
Role of PolyP in Bacteria
Kornberg A., N.N. Rao, D. Ault-Riche. Inorganic Polyphosphate: A molecule of Many Functions. Annu. Rev. Biochem. 1999: 68: 89-125.
Role of PolyP in Archaea
•  Cloned, sequenced and overexpressed PPX from
S. solfataricus
•  Purified recombinant PPX showed high activity
with long-chain polyP at 50-60 ºC
•  PPX shows highest similarity (25-45%) to
sequences of the bacterial PPXs
•  First report of enzyme in archaea to be involved in
polyP metabolism.
Cardona, ST, FP Chavez, CA Jerez. The Exopolyphosphatase Gene from Sulfolobus solfataricus: Characterization
of the First Gene Found To Be Involved in Polyphosphate Metabolism in Archaea. AEM. 2002. 4812-4819.
Thermoacidophiles
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Found in hot springs or deep ocean vents
Grows pH range 2-4
Temperature range optimum 75°C-85°C
Important for Biomining
Sulfolobales
Yellowstone National Park courtesy of Dr. Ken Stedman .
microbewiki.kenyon.edu/images/thumb/c/ce/Sulf..
Metal Tolerance in Acidophiles
Dopson, M. C. Baker-Austin, P.R. Koppineedi, P.L. Bond. Growth in sulfidic mineral environments: metal resistance mechanisms in
Acidophilic micro-organisms. Microbiology. 2003: 149: 1959-1970.
Mechanisms of metal and toxin
resistance in bacteria
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Exopolysaccharide production
Detoxification thru efflux system
Metal sequestration
Tight coupling between membrane transport
metal efflux proteins/ATPases
•  Enzymatic transformations
Dopson, M. C. Baker-Austin, P.R. Koppineedi, P.L. Bond. Growth in sulfidic
mineral environments: metal resistance mechanisms in Acidophilic microorganisms. Microbiology. 2003: 149: 1959-1970.
Proposed Mechanisms of Copper Tolerance
Enterococcus hirae
Silver & Phung. Bacterial Heavy Metal Resistances.
Annu. Rev. Microbiol. 1996. 50: 753-789.
Pseudomonas syringae
S. solfataricus CopB
Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase From
the thermoacidophilic Archaeon Sulfolobus solfataricus. Journal of Bioenergetics and Biomembranes. 2004. 36:151-159.
Questions:
  What is the role of polyP in Archaea?
  What role does it play in metal tolerance
in Archaea?
  Does it fit the proposed metal tolerance
model?
Fig. 1
Full line: S. metallicus
Dashed: S. acidocaldarius
Dotted: hydroxyapatite
S. solfataricus
S. metallicus
Fig. 2
polyP levels
cell numbers
S. metallicus
S. solfataricus
S. acidocaldarius
Fig. 3
+ CuSO4
Absent of metal
S. metallicus
S. solfataricus
Absent of metal
Fig. 4
Absent CuSO4
S. metallicus
100mM
200mM
Fig. 5
PPX response to CuSO4 in vitro
CuSO4
MnSO4
S. metallicus
Fig. 6
PPX activity and PolyP content
100mM
20
10
None
None
10
20
100mM
S. metallicus
Fig. 7
Efflux of Pi from S. metallicus cells
200mM
100mM
None
Conclusions
  Do the results fit with the proposed
metal tolerance model?
Questions….
Polyphosphate kinase PPK
Kornberg A., N.N. Rao, D. Ault-Riche. Inorganic Polyphosphate: A molecule of Many Functions. Annu. Rev. Biochem. 1999: 68: 89-125.
Copper Resistance Mechanisms
  Intracellular complexation
  Decreased accumulation
  Extracellular complexation
  Sequestration in periplasm