1. Microbial Diversity Gram-Negative Strategies for Survival Nitrosomonas vs. Pseudomonas Specialist vs. Jack-of-all-Trades

2. 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. NH 3 NO 2 NO 3 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 CO 2 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 10 6 bp overall G+C content = 50.8%

3. 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 10 6 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.

4. Question?? Which of these microbes would you choose as a candidate for potential new natural products or activities? Nitrosomonas vs. Pseudomonas

5. Sequence map of Pseudomonas aeruginosa PAO1

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

8. Gene function classes

9. 1223072 3792289 3889743

10. 0.1 Microbacterium Sp PCOB-2 Halobacterium Flavobacterium sp36 Sulfolobus B. subtilis Agrobacterium Stenotrophomonas Burkholderia Acinetobacter P. putida Halomicrobium P. aeruginosa Klebsiella Serratia Alcanivorax Marinobacter Acidithiobacillus Actinoplanes Arthrobacter Corynebacterium Gordonia Mycobacterium Nocardia Rhodococcus Brevibacillus Streptococcus Lactobacillus B. licheniformis B. megaterium 16s rDNA tree of biosurfactant-producing bacteria

11. 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………… A Problem:

12. O-CH- CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 3 C-CH 2 -CH - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 2 - CH 3 O -O-O O HO OH OR CH 3 O Rhamnolipid monomer Micelle formation at >CMC concentrations CMC = 0.1 mM (50mg/L) O=C CH 2 Ca 2+ 5 nm diameter Pseudomonas in bioremediation Pseudomonas aeruginosa makes a surfactant molecule, rhamnolipid:

13. 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

14. 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)

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

16. 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+/-

17. 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

18. The reason for variable results is that surfactants are usually added at high concentration (>> CMC), wherein they can: Brazito soil be toxic to degrading cells serve as a preferred carbon source

20. What are biosurfactant effects on cell surface? Biosurfactant removes LPS from outer membrane of Gram-negative cells Cell surface becomes more hydrophobic

22. Cell Surface Hydrophobicity

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

25. 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?

26. Effect of rhamnolipid on MIC of hydrophobic antibiotics

27. Effect of rhamnolipid on dye (DMP) uptake DMP = 2-(4-dimethylaminostyryl) 1-ethylpyridinium

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

29. 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.

30. Question: How will we develop the tools necessary for systematic discovery of new natural products and other activities?