Why does the Gene 2 mutant become an auxotroph and also lose nitrogen-fixing ability?

Representative abundances of selected metabolites found in a small molecule extract (SME) from your designated wild-type bacterial strain are shown in the table below. Note: These reported abundances are dependent on the solubility and ionization characteristics of each compound within this complex mixture. They, therefore, have no direct connection with the metabolite concentrations in the cell.

Metabolite

Abundance in wild-type (mM)

Citrate

0.04

Isocitrate

0.04

2-oxoglutarate

8.1

Succinyl CoA

470.0

Succinate

480.0

Fumarate

300

Malate

0.5

Oxaloacetate

350

A number of mutants with a defective TCA cycle enzyme have been isolated. You prepared a small molecule extract from ONE mutant that has been identified in the gene encoding the enzyme malate dehydrogenase.

Q2.3. Identify the metabolites expected to change drastically in the mutant and indicate whether you expect the concentration to increase or decrease (3 marks).

The IMG search showed that there are two genes that code for a specific TCA cycle enzyme in this strain. You used a targeted method to create a point mutation in each gene. The phenotypes of the mutant are presented in the following table.

Mutant

Growth requirement (in a free-living environment)

Nitrogen fixation (symbiotic condition)

Gene 1 mutant

Prototroph

Fix+

Gene 2 mutant

Auxotroph

Fix-

Q2.4. Why does the Gene 1 mutant remain prototrophic and nitrogen-fixing?

Q2.5. Why does the Gene 2 mutant become an auxotroph and also lose nitrogen-fixing ability?

Q2.6. Can you predict the conditions/environment where (a) gene 1, and (b) gene 2 are expressed?

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