General Microbiology - Schlegel H. 1987

Regulation of Metabolism
Mutants with Impaired Regulation

Elucidating the mechanisms that regulate enzyme Biosynthesis and activity has been made possible through the isolation of regulatory mutants. Several types of mutants have been isolated, including: 1) those that fail to form a functionally competent repressor protein or contain it in vastly elevated amounts; 2) those with a constitutive operator that is unable to bind the repressor protein; and 3) those exhibiting allosteric insensitivity, where a specific enzyme fails to recognize its effector. Below, we describe some of the Methods used to isolate such mutants.

Mutants constitutively synthesizing catabolic Enzymes. Enrichment cultures of such mutants can be obtained through repeated shifts in substrates. If Cells constitutively synthesize the enzymes required to utilize substrate A, they immediately resume growth at a maximum rate upon transfer of The Cell population from substrate B to substrate A; by contrast, inducible wild-type cells require a distinct lag phase to synthesize the enzymes needed for growth on substrate A. After several generations, the cells are returned to the medium containing substrate B and allowed to grow until the enzymes involved in substrate A utilization are sufficiently 'diluted out'. Repeating this Procedure multiple times allows the constitutive mutants to strongly outgrow the wild-type cells with inducible enzymes. This approach has been used, for example, to isolate E. coli mutants that constitutively synthesize the enzymes required for lactose utilization. Other Selection methods rely on repressing induction using structural substrate analogues. For instance, methylthiogalactoside can suppress the galactose-Induced Expression of the gal Operon in Escherichia coli.

Mutants constitutively synthesizing anabolic enzymes. These mutants, as well as those with defects in fine-tuned regulatory processes of biosynthesis, can be isolated using antimetabolites. Many antimetabolites (Section 6.6), acting as structural analogues of normal biosynthetic end products (Amino Acids, Pyrimidines, etc.), exert a bacteriostatic effect. Mimicking the end product, they disrupt the synthesis of normal metabolites on the one hand, and become incorporated into Proteins or Nucleic Acids on the other, resulting in macromolecules incapable of functioning normally. Inhibition by such a 'false' end product halts growth. If a wild-type population (10⁸–10¹⁰ cells) is plated onto an Agar medium containing an antimetabolite, only rare resistant mutants will retain The ability to grow and form colonies.

Such antimetabolite resistance can stem from various mutation-induced alterations in the physiological Properties of the cell. Let us examine the types of such Mutations.

1. Mutations leading to 'allosteric insensitivity'. In this type of mutation, neither the metabolite nor the antimetabolite can inhibit The activity of the first (allosteric) enzyme in the biosynthetic pathway. As a result, The formation of the corresponding end product is left unregulated.

2. Mutations leading to constitutive derepression. The consequence of this mutation is the uncontrolled Synthesis of the enzymes involved in producing the end product.

3. Mutations affecting the catalytic centers of enzymes that activate metabolites and participate in their conversions. By increasing its selectivity, the enzyme may lose the ability to bind the antimetabolite in place of the metabolite, rendering the antimetabolite no longer bacteriostatic.

4. Mutations disrupting transport processes. As a result of such mutations, antimetabolites can no longer enter the cell and therefore can no longer interfere with its METABOLISM.

5. Mutations causing the constitutive degradation of antimetabolites. Here, the cell breaks down the antimetabolite, thereby detoxifying it. From the standpoint of selecting regulatory mutants, only the first two Types of mutations are of interest. Derepression of anabolic enzyme synthesis and the loss of sensitivity to allosteric inhibition often lead to the 'overproduction' and excretion of the pathway's end product (metabolite) into the medium. This is advantageous for the mutant cell because the metabolite displaces the antimetabolite from the reaction, thereby enabling cell growth and colony formation. The excess metabolite produced is secreted, diffuses into the agar, and neutralizes The Effect of the antimetabolite on wild-type cells within the diffusion zone. These cells begin to grow and form small colonies, referred to as secondary or satellite colonies. The central colony, meanwhile, is formed by the mutant cells excreting the metabolite (Fig. 16.15). The growth of satellites indicates that a mutation has occurred disrupting normal regulatory mechanisms. However, determining the specific defect responsible for the accumulation and excretion of the metabolite requires dedicated analysis in each case.

The antimetabolite-based approach has successfully yielded numerous regulatory mutants. Comparing various mutants has revealed that the loss of the repression mechanism has less impact on The rate of end product synthesis than does the alteration of allosteric inhibition. In mutants incapable of feedback inhibition along a specific biosynthetic pathway, the end product accumulates within the cell and is frequently excreted into the medium, despite completely normal repression. Conversely, mutants exhibiting strong derepression (constitutivity) show only very minor accumulation and excretion of the end product if it exerts a normal inhibitory effect. Thus, repression is primarily important for minimizing the metabolic costs associated with mRNA and Protein Synthesis, whereas the synthesis of metabolites is regulated primarily through end-product inhibition.

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Fig. 16.15. Mutants resistant to an antimetabolite. Appearance of a Petri dish containing a nutrient agar medium supplemented with an antimetabolite. The agar surface was inoculated with Bacteria (approximately 10⁹ cells). Initially, only resistant mutants formed colonies. The halos of secondary colonies surrounding the two large colonies indicate that the Cells of the latter are excreting a metabolite.

Mutants with altered effector sensitivity. Mutants in which the effector sensitivity of an allosteric enzyme has been altered can also be isolated using an entirely different strategy, namely as revertants to auxotrophy. The procedure is as follows. First, regulatory mutants are isolated that are auxotrophic for the metabolite intended for recovery as an end product accumulating in the medium. Next, among these auxotrophic mutants, those whose inability to synthesize the given metabolite stems from a defect in an allosteric enzyme of the corresponding biosynthetic pathway are selected. Prototrophic revertants are then isolated from this mutant population; these no longer require the end product because they have regained the ability to synthesize it themselves. Among the revertants, those that excrete the desired product into the medium are selected. They can be detected using the bioautographic method (Section 10.2.2) or identified by the growth of satellite colonies. Such a mutant, obtained through a two-step selection process, can be conceptualized as follows: the first mutation disabled the catalytic center of one of the allosteric enzymes. The second mutation altered the Structure (conformation) of the entire protein molecule, restoring the catalytic activity of the enzyme while abolishing its allosteric sensitivity. In this case, as in many others, isolating the desired mutant requires a series of steps involving mutagenesis and selection.

Theoretical and applied aspects. The strategy for mutant selection is crucial for advancing the Study of Cellular metabolism and elucidating regulatory mechanisms. At the same time, this strategy holds significant practical value, as it dictates the approaches for the targeted selection of high-yield producers for all substances obtainable via microorganisms.