The order of a biosynthetic pathway with diffusible intermediates can often be determined by crossfeeding experiments. Consider the following biosynthetic pathway:
A mutation in either gene 1 or gene 2 can prevent synthesis of Z. In cells with a mutation in gene 2 intermediate Y may accumulate and ultimately be excreted into the surrounding medium (because Y is not being consumed to make Z in this mutant). In cells with a mutation in gene 1 cannot make Y, since gene 2 is functional they can convert exogenous Y into Z. That is, the Y excreted by gene 2 mutants can crossfeed the gene 1 mutants.
For example, the following cartoon shows the growth of tryptophan auxotrophs on a minimal medium plate supplemented with a tiny amount of tryptophan.
All three mutants grow until the tryptophan is exhausted, resulting in faint growth where the cells had been streaked. However, in the area where the trpB and trpD cells were close to each other, the trpD mutants grew much better, indicating that the trpB mutants excrete a product that the trpD mutant can convert to tryptophan. Hence, the metabolic block in the trpB mutant must be at a later step than in the trpD mutant. Likewise, the metabolic block in the trpD mutant must be at a later step than in the trpE mutant. Thus, the conclusion is that the three gene products act in the following order:
If the intermediates are available, it is also possible to simply test to see which of the mutants can use which intermediates. This can be done in liquid medium containing the intermediates or on agar plates containing the intermediates. A very simple way of checking for growth on different intermediates is called a crystal test. The mutant is spread on a minimal agar plate lacking the auxotrophic requirement, and a few crystals of each intermediate are added near the edge of the plate -- growth near the crystals indicates that the mutant can use that intermediate.
The crossfeeding results were confirmed by testing growth with specific biochemical intermediates as shown in the table below.
|Mutation||Growth on minimal medium plus:||Intermediate|
Some question to ponder:
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Last modified February 14, 2000