Bacteria in the BBB Ponds In this project, we plan to investigate the bacteria in the BBB ponds and differences in their photosynthetic light-harvesting centers. Generally, light harvesting centers and their satellite complexes are composed of bacteriochlorophylls and carotenes. We will identify these pigments in several different cultures, including in purple sulfur bacteria (Chromatium spp) and green sulfur bacteria (Chlorobium spp) using characteristic absorbance values and thin layer chromatography. Comparing the Caltech strains with different pigments may allow us to estimate and understand differences in energy yield. Along the way, we will also be using mixed and single culture methods to isolate bacteria and investigating the plate count anomaly using a variety of media. “Species” on each plate will be categorized based on gross colony morphology and compared to plates ofother media and to the Winogradsky column, a mixed culture method.Timeline:Jan 28 - Pond field trip- talk/discuss- Make Winogradsky column- mixed culture- Start plate count anomaly (mixed culture pure culture)Feb 2 - Retreat (Background reading)Feb 4 - Retreat (Background reading)Feb 9 - Select colorful colonies for streaking- Grow purple sulfur bacteria (ATCC Medium 37, infrared lights?) Use Winogradsky for inoculumFeb 11 - Examine specimens from pure streaked culture with microscope - Plate duplication: o Broth or LB pond, minimal, min+carrot - Compare later with survival on new media.Feb 16 - Biodiversity calcFeb 18 - Scan absorbances of Bacteria--??o like Overmann Paper- compare with his. Feb 23 - Use purple cultures and broth cultures on TLC to separate pigments; try to identify carotenoids, etc…Feb 25 - Progress Report due- Bulk doubling time exptsMar 2 - Look at bacteria from Winogradsky column with microscopeo Analyze: Same as from plate count anomaly?Mar 4 - Discuss QM of photosynthesis; estimate efficiencies of strains with different pigments based on doubling timeMar 9 - Work on Presentations, practice talkMar 11 - PresentationsMaterials:Winogradsky column: Mixed culture in a clear column or jar. (See “A Field Guide to Bacteria” by Betsey Dexter Dyer.)Pure culture: Growing a single strain of bacteria in liquid or on agar plates. ATCC Medium 37 (for Chromatium species): See http://www.atcc.org/Attachments/3682.pdf This includes vitamin B12, which is necessary for rapid growth in Chromatium. For plates, add 15 g agar per L of media. Autoclave, let cool. Add 1 mL Vit B 12 solution perliter, mix and pour.Chlorobium Media: (1 L)0.30 g KH2PO40.34 g NH4Cl0.34 g KCl0.15 g CaCl2*2H2O0.50 g MgSO4*7H2O 1.00 mL Trace solutionAutoclave, cool, and add: 15.0 ml 10% (w/v) NaHCO3 (saturated with CO2)6.0 ml 10% (w/v) Na2S*9H2O (autoclaved)1.0 ml Vitamin B12 solution (2 mg B12 in 100 ml distilled water)Adjust pH to 6.7 with sterile 2 M H2SO4. Dispense into 100-ml bottles.Trace solution (1-liter)2.00 g FeSO4*7H2O – dissolve in 10 ml HCl (25% w/v); add DI water and:190 mg CoCl2*6H2O100 mg MnCl2*4H2O70 mg ZnCl236 mg Na2MoO4*2H2O24 mg NiCl2*6H2O6 mg H3BO32 mg CuCl2*2H2OFor plates, add 15 g agar per L of media. Autoclave, let cool. Add 1mL Vit B12 solution mix and pour.1. Minimal Media: To make M9 Salts aliquot 800ml H2O and add:% 64g Na2HPO4-7H2O% 15g KH2PO4% 2.5g NaCl% 5.0g NH4Cl% Stir until dissolved% Adjust to 1000ml with distilled H2O% Sterilize by autoclaving2. Measure ~700ml of distilled H2O (sterile) and add 200ml of M9 salts, 2ml of 1M MgSO4 (sterile), 20 ml of 20% glucose (or other carbon source), 100ul of 1M CaCl2 (sterile) and adjust to 1000ml with distilled H2O. Pond Water Media: Filter sterilize 500 mL pond water. (add 15 g agar per L for plates, autoclave and pour.)Chicken/Beef Bouillon Media: Dissolve 1 11g cube (Knorr) in 1L DDH2O. Autoclave. Skim off fat, and autoclave again. For plates, add 15 g agar per L before second autoclaving.(Alt.) Dissolve 1 cube in 2 L DDH2O. (Clearer, easier to see.)Bibliography:Andersson et al. Femtosecond dynamics of carotenoid-to-bacteriochlorophyll a energy transfer in the light-harvesting antenna complexes from the purple bacterium Chromatium purpuratum. Chemical Physics (1995) vol. 210 pp. 195-217Delves into QM of photosynthesis.Damjanovic et al. Energy transfer between carotenoids and bacteriochlorophylls in light-harvesting complex II of purple bacteria. (1999) vol. 59 (3) pp. 3293-3311Explains pretty well how the light harvesting centers are organized and explains electron transfer.Devereux et al. Natural Relationships among Sulfate-Reducing Eubacteria. Journal of Bacteriology (1989) pp. 1-716s sequences used to compare Desulfos.Franz et al. Utilization of 'elemental' sulfur by different phototrophic sulfur bacteria (Chromatiaceae, Ectothiorhodospiraceae): A sulfur K-edge XANES spectroscopy study. J. Phys.: Conf. Ser. (2009) vol. 190 pp. 012200Structure of carotenoids and what we can learn through structural studies.Fujii et al. 1 H NMR, electronic-absorption and resonance-Raman spectra of isomeric okenone as compared with those of isomeric i -carotene, canthaxanthin, i -apo-8'-carotenal and spheroidene. Spectrochimica Acta, Part A (1998) vol. 54 pp. 727-743More Carotene StructureImhoff. The Chromatiaceae. Prokaryotes (2006) (6) pp. 846-873A good background on Chromatia spp., the purple sulfur bacteria. This is an excellent place to start.Leadbetter. Cultivation of recalcitrant microbes: cells are alive, well and revealing their secrets in the 21st century laboratory. Current Opinion in Microbiology (2003) vol. 6 pp. 274-281Background on how we can use selective enrichment to grow bacteria we’re looking for. Overmann and Glaeser. Selective enrichment and characterization of Roseospirillum parvum, gen. nov. and sp. nov., a new purple non-sulfur bacterium with unusual light absorption properties. Arch Microbiol (1999) vol. 171 pp. 405-416Selective enrichment with light instead of media.Reinhartz et al. Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum. Arch Microbiol (1998) vol. 170 pp. 59-68How Chromatium reduce sulfur. Toropygina et al. Reconstitution of Okenone into Light Harvesting Complexes from Allochromatium minutissium. Biochemistry (Moscow) (2005) vol. 70 (11) pp. 1231-1237Some groups use Chromatium as a model system for bacterial photosynthesis. Here they trade the normal carotene for high-yield okenone.Whitman et al. Prokaryotes: The unseen majority. PNAS (1998) pp. 6578-6583A neat