ATP synthase (ATPase, Complex V) is a highly conserved protein which converts pmf into ATP; it catalyzes a reversible reaction between ATP and ADP + P_i. It has 2 parts: F₁ is a multi-unit piece on the cytoplasmic side, and F_o spans the membrane and is the proton-conducting channel.

• NADH and FADH₂ are e^- carriers. NADH carries more e^- than FADH₂.
• Nitrogen fixation cofactor, MoFe, can change oxidation states. MoFe gets e^- from an Fe^- protein called dinitrogenase. Haber Process: N₂^-→NH₃ at 400ºC, 200 atm and Fe^-catalyst.
• Anaerobically, SO₄^2- or NO₃^- are terminal e^- acceptors instead of O₂.
• Denitrification reduces NO₃^- into N₂.
• Chemolithotrophs can oxidize inorganic compounds for energy.
• Chl is light-sensitive, MG-containing porphyrin initiating photophosphorylation.

Auxotrophs are unable to synthesize all their own biomolecules (nutrients, such as amino acids). For example, a bio^- mutant is unable to synthesize biotin. Auxotrophs can be studied to understand pathways for the synthesis of amino acids. Also, they can be used for other experiments (such as the Ames test and R-factor experiment).

Isolating Auxotrophs

Isolating a lysine auxotroph, from a mixture of auxotrophs and prototrophs, is a simple procedure. You can easily modify this procedure to isolate almost any specific kind of auxotroph.

1. Grow cells in media containing penicillin (which will kill dividing cells) and all amino acids except lysine.
2. Remove the penicillin-laced media and replace with media containing lysine
3. Screen for lysine auxotrophs
4. Further assays may be performed to determine the location of the mutation in the lysine synthesis pathway

Another, more efficient procedure is to:

1. Plate auxotroph/prototroph mixture on enriched GMA
2. Screen for tiny colonies
3. Pick tiniest colonies
4. Grow on GMA and YTA
5. Colonies able to grow only on YTA are auxotrophs

Auxotrophs form tiny colonies because there is only a small amount of complex media in enriched GMA. This is an example of screening, because you distinguish between the two different colony types (auxotrophs and prototrophs) based on colony morphology.

However, if you want to isolate any cell exhibiting auxotrophy (from a batch of overwise prototrophic cells) then the procedure is different. This can be helpful if you want to determine the pathway for synthesizing a certain compound. The procedure for outlining the biosynthetic pathway for lysine is given below. You will use E. coli as your start culture because WT E. coli are prototrophic.

1. Inoculate minimal broth with E. coli
2. Mutagenize culture, resulting in mixture of auxotrophs and prototrophs
3. Add penicillin
4. Replace media with minimal broth + glucose + lysine
5. Plate onto enriched GMA
6. Pick tiny colonies, inoculate GMA and GMA+lysine
7. Colonies that grow only on GMA+lysine are lysine auxotrophs
8. Perform a —- test.