Microbial Diversity

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Microbial Diversity Gram-Negative Strategies for
Survival
Nitrosomonas vs.
Pseudomonas Specialist vs.
Jack-of-all-Trades
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• Nitrosomonas europaea
• Gram-negative, chemoautotroph
• Specializes in ammonia oxidation. These bacteria
  are important in
• the treatment of industrial and sewage waste in
  the first step of
• oxidizing ammonia to nitrate.
• NH3 NO2 NO3
• Found in soil, freshwater, sewage, the walls of
  buildings and on the
• surface of monuments especially in polluted
  areas where air contains
• high levels of nitrogen compounds.
• Problematic because can reduce availability of
  nitrogen to plants and
• hence limit CO2 fixation. Also may contribute
  significantly to the global
• production of nitrous oxide.
• N. europaea strain Schmidt Stan Watson is now
  completely sequenced.

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• Pseudomonas aeruginosa
• Gram-negative, chemoheterotroph
• Versatile
• Found in soil, marshes, coastal marine habitats,
  
• on plants and animals
• Problematic for cystic fibrosis, burn victims,
• cancer, ICU patients
• P. aeruginosa PAO1 is now completely sequenced.
  
• - 5570 predicted genes
• - 6.3 x 106 bp (largest sequenced genome to
  date)
• - overall GC content 66.6
• - isolated regions with lower GC content may
  be result of recent
• horizontal gene transfer

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Question??
Which of these microbes would you choose as a
candidate for potential new natural products or
activities?
Nitrosomonas vs. Pseudomonas
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Sequence map of Pseudomonas aeruginosa PAO1
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Sequence map of Pseudomonas aeruginosa PAO1
phe genes
rhl genes
We can use these maps to gather information on
genes of interest.
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Gene function classes
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1223072
3792289
3889743
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A Problem

• Despite the fact that we appreciate the great
  diversity represented by the eubacterial kingdom,
  we currently have no way to translate this into
  interpreting associated activities.
• Therefore, discovery of new natural products is
  inhibited
• we do not have adequate discovery tools
• - we cannot interpret new gene sequences
• - we do not have the capability to isolate
  unique
• molecules from the environment at the levels
  normally
• produced.
• Yet the potential for new and economically
  important discoveries is extremely high.
• An example

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Pseudomonas in bioremediation
Pseudomonas aeruginosa makes a surfactant
molecule, rhamnolipid
O
C-CH2-CH - CH2 - CH2 - CH2 - CH2 - CH2 - CH2 - CH3
-O
O
Ca2
OC
CH2
O
HO
CH3
O-CH- CH2 - CH2 - CH2 - CH2 - CH2 - CH2 - CH3
OH OR
Rhamnolipid monomer
Micelle formation at gtCMC concentrations CMC
0.1 mM (50mg/L)
5 nm diameter
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Synthetic Surfactant Industry Trivia

• Estimated business of over 9 billion/yr
• Cost 1 to 5/kg
• There is a Surfactant Science Series containing
• gt 50 volumes

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Surfactant Industry

• Agriculture
• Cosmetics
• Textile processing
• Detergents (industry, home)
• Building and construction (paving and concrete
  additive)
• Metal Processing (e.g., ore concentration, rust
  removal)
• Polymers (emulsion stabilizers, plasticizers)
• Paint and protective coatings
• Paper (resin removal, washing)
• Petroleum production and products
• Leather processing
• Pharmaceuticals
• Food (fat emulsifier, sugar processing,
  solubilization of
• flavor oils)

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• Rhamnolipid Applications
• Production of fine chemicals
• Bioremediation
• biodegradation of organics
• biodegradation in the presence of toxic metals
• removal of organics by flushing
• removal of metals by flushing
• Biological control
• Antibiotic facilitator

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• Literature reports show variable effects
• of surfactants on biodegradation
• Aiba et al. (1969) synthetic surfactant -
• Aronstein and Alexander (1992) synthetic
  surfactant
• Breuil and Kushner (1980) synthetic
  surfactant /-
• Churchill et al. (1995) synthetic surfactant
• biosurfactant
• Falatko and Novak (1992) biosurfactant /-
• Graves and Leavitt (1991) synthetic
  surfactant -
• Jain et al. (1992) biosurfactant
• Oberbremer et al. (1990) biosurfactant
• Providenti et al. (1995) biosurfactant /-
• Thiem (1994) synthetic surfactant /-
• Whitworth et al. (1973) synthetic surfactant
• Zhang and Miller (19941995) biosurfactant /-

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• 1. Bioremediation of organic contaminants
• Rates are constrained by low bioavailability
• sorption
• low aqueous solubility
• aging
• Biosurfactants increase bioavailability
• solubilization
• alteration of cell surface properties

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The reason for variable results is that
surfactants are usually added at high
concentration (gtgt CMC), wherein they can
be toxic to degrading cells
serve as a preferred carbon source
Brazito soil
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• What are biosurfactant effects on cell surface?
• Biosurfactant removes LPS from outer membrane
  of Gram-negative cells
• Cell surface becomes more hydrophobic

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Cell Surface Hydrophobicity
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Conclusion
Biosurfactants may be most effective at low
concentration for remediation of organic
contaminants.
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• 2. Facilitating antibiotic activity
• Antibiotic resistance is a serious and widespread
  problem. There are several reasons for
  antibiotic resistance. One that is particularly
  important for hydrophobic antibiotics is that
  cells do not take up the antibiotic.
• Can rhamnolipid be used to facilitate the uptake
  of hydrophobic antibiotics?

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Effect of rhamnolipid on MIC of hydrophobic
antibiotics
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Effect of rhamnolipid on dye (DMP) uptake
DMP 2-(4-dimethylaminostyryl) 1-ethylpyridinium
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Effect of rhamnolipid on the detergent-induced
lysis of P. aeruginosa.
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Conclusions
These results suggest that a small amphipathic
molecule, like rhamnolipid, could be used in
combination with an antibiotic as a therapy to
decrease antibiotic dosage and increase
antibiotic efficacy in a patient with a
gram-negative infection.
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Question How will we develop the tools
necessary for systematic discovery of new natural
products and other activities?