Microbial Diversity Gram-Negative Strategies for Survival - PowerPoint PPT Presentation

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Microbial Diversity

Gram-Negative Strategies for Survival

Nitrosomonas vs. Pseudomonas

Specialist vs. Jack-of-all-Trades

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.
2715 predicted genes, 2.80 x 106 bp
overall G+C content = 50.8%

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 G+C content = 66.6%
- isolated regions with lower G+C content may be result of recent
horizontal gene transfer
- > 500 genes are transcriptional regulators or environmental
sensors. Has more than twice the number of two-component
regulators than E. coli or B. subtilis.

Question??

Which of these microbes would you choose as a candidate for potential new natural products or activities?

Nitrosomonas vs. Pseudomonas

Sequence map of Pseudomonas aeruginosa PAO1

phe genes

rhl genes

We can use these maps to gather information on genes of interest.

Gene function classes

1223072

3792289

3889743

16s rDNA tree of biosurfactant-producing bacteria

Sulfolobus

Acinetobacter

P. putida

P. aeruginosa

Burkholderia

Halomicrobium

Stenotrophomonas

Alcanivorax

Agrobacterium

Marinobacter

Klebsiella

Serratia

Acidithiobacillus

Actinoplanes

Arthrobacter

Halobacterium

Microbacterium

Sp PCOB-2

Corynebacterium

Gordonia

Flavobacterium sp36

Rhodococcus

Nocardia

Streptococcus

Lactobacillus

Brevibacillus

Mycobacterium

0.1

B. subtilis

B. licheniformis

B. megaterium

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…………

Pseudomonas in bioremediation

Pseudomonas aeruginosa makes a surfactant molecule, rhamnolipid:

O

C-CH2-CH - CH2 - CH2 - CH2 - CH2 - CH2 - CH2 - CH3

-O

O

Ca2+

O=C

CH2

O

HO

CH3

O-CH- CH2 - CH2 - CH2 - CH2 - CH2 - CH2 - CH3

OH OR

Rhamnolipid monomer

Micelle formation at >CMC concentrations

CMC = 0.1 mM (50mg/L)

5 nm diameter

Synthetic Surfactant Industry Trivia

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

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)

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

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 (1994;1995) biosurfactant +/-

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

The reason for variable results is that surfactants are usually added at high concentration (>> CMC), wherein they can:

be toxic to degrading cells

serve as a

preferred

carbon

source

Brazito soil

What are biosurfactant effects on cell surface?

Biosurfactant removes LPS from outer membrane of Gram-negative cells
Cell surface becomes more hydrophobic

Cell Surface Hydrophobicity

Conclusion

Biosurfactants may be most effective at low concentration for remediation of organic contaminants.

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?

Effect of rhamnolipid on MIC of hydrophobic antibiotics

Effect of rhamnolipid on dye (DMP) uptake

DMP = 2-(4-dimethylaminostyryl) 1-ethylpyridinium

Effect of rhamnolipid on the detergent-induced lysis of P. aeruginosa.

Conclusions

These results suggest that asmall 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.