General Microbiology - Schlegel, H. 1987

Constancy, variation, and transmission of traits
Mutations and their origin

15.2.1 The Non-directed Nature of Mutations

The concept that abrupt changes in hereditary traits—mutations—can also occur in microorganisms gained acceptance only with great difficulty. Before The Development of pure culture techniques, many scientists (Nägeli, Zopf) believed that the Morphology and physiological properties of Bacteria were extremely variable. It was widely thought that the numerous bacteria found in nature simply represented different life-cycle stages of a small number of species (pleomorphism). Arguing against this view based on results obtained with improved techniques and pure cultures, other researchers championed the monomorphic theory, which held that bacteria could be distinguished and classified based on the constancy of their morphological and physiological characteristics. It became necessary to learn how to distinguish between the genotype and phenotype in bacteria as well. The genotype refers to the total set of hereditary determinants of a Cell, in contrast to the phenotype, which is the sum of its observable traits. The phenotypic expression of the same genotype can vary depending on environmental conditions.

The term mutation was introduced by de Vries, who studied Variability and heredity in plants and defined a mutation as "an abrupt change in a heritable trait." This concept was later extended to bacteria by Beijerinck.

In bacteria, variants arising under METABOLISM/18.html">The Influence of poisons attracted the most attention. For a long time, these poison-resistant Cells were considered the result of adaptation. Distinguishing phenotypic adaptation to altered environmental conditions from genotypic change proved to be no easy task, although the distinguishing criterion is quite simple. Phenotypic adaptation occurs in all cells of a given culture, whereas a genotypic change affects only a few cells; owing to their enhanced fitness for the environment, cells that have become hereditarily resistant—for instance, to a toxin—grow faster than the Cells of the parent strain and eventually outcompete them.

Another question arose: Are genotypic changes caused by the selective factor itself exerting a directed influence on the genes? Or do mutations occur independently of the environment and are they non-directed in nature? Lamarck's theory of the inheritance of acquired adaptive traits turned out to be untenable for higher organisms. Darwin's theory gained widespread acceptance; according to this view, new types and species arise through environment-independent mutations, followed by the Selection of the fittest forms. In higher organisms, mutations that are passed on to offspring occur in Germ Cells, which are largely shielded from environmental influences. In contrast, bacterial cells are readily exposed to external factors. Consequently, one might imagine that the action of a toxin leading to the appearance of resistant mutants in a bacterial population is not restricted to mere selection alone, but also determines the direction of the mutations. The question of whether mutations depend on the environment and whether they are directed remained a pivotal problem in biology and had to be resolved using bacteria.

As a result of a series of highly influential experiments, evidence was obtained that mutations in bacteria are likewise spontaneous and non-directed. Here, we will consider only one of these classic experiments; it is easy to understand and illustrates the widely used replica plating technique.

Indirect selection of mutants by replica plating. This ingenious method, which is now widely used, was developed by the Lederbergs in 1952. It works as follows. A velvet-covered stamp, slightly smaller in diameter than a Petri dish, is pressed onto an Agar plate on which bacterial colonies are growing. A fraction of the cells adheres to the velvet nap. If this stamp is then pressed onto an uninoculated agar plate, the entire pattern of colonies from the original plate is reproduced on the new plate.

From plate I, which had a dense lawn of phage-sensitive bacteria (Fig. 15.2), cells were transferred using the velvet stamp onto plain nutrient agar (II) and onto agar previously seeded with phage (III). After incubation, isolated phage-resistant colonies appeared. Subsequently, a sample was taken from plate II (plain agar) at a Location corresponding to the area of the phage-resistant colonies on the phage plate. The proliferated cells were spread over the agar in a new dish and, after incubation, transferred using the velvet stamp onto two plates (plain agar and phage-seeded agar). This Procedure was repeated many times, and ultimately a suspension of phage-resistant mutants was obtained that had never had any contact with the phage. This experiment provides clear proof of the spontaneous emergence of phage-resistant mutants without any prior contact with the selective agent.

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Fig. 15.2. Proof of the non-directed nature of bacterial mutations using the replica plating technique. See text for explanation.

15.2.2 Spontaneous and Induced Mutations

Spontaneous Mutations

Mutations regularly arise within a bacterial population without any experimental intervention; such mutations are called spontaneous mutations, and the cells in which they occur are termed spontaneous mutants. The mutagenic effect of DNA base analogs (see below) points to the likely causes of spontaneous mutations: presumably, these involve random errors during nucleotide incorporation during DNA Replication—errors caused by tautomeric proton shifts in the bases. Thymine, for example, normally exists in the oxo form, in which it forms Hydrogen Bonds with adenine. However, if thymine tautomerizes to the enol form during base pairing in DNA replication, it pairs with guanine instead. As a result, a G—C pair appears in the new DNA molecule at the site previously occupied by an A—T pair.

Mutant proportion in the population and mutation rate. The numerical proportion of mutants in a cell population varies for different traits, typically ranging from 10^-4 to 10^-11. It depends on the mutation rate, environmental conditions, the age of The Cell suspension, and other factors. The probability of specific mutations occurring per cell per generation is called the mutation rate. At high growth rates, it is constant and is usually determined for cells in the exponential growth phase under optimal environmental conditions. The spontaneous mutation rate for a given Gene is on the order of 10^-5, and for a specific nucleotide pair, about 10^-8.

"Silent" mutations. While mutation in the traditional sense is understood as a sudden phenotypic change in a trait—that is, a genotypic alteration manifested in the phenotype—at THE MOLECULAR LEVEL, any stable, heritable change in DNA is considered a mutation. However, due to the degeneracy of The Genetic Code, it is clear that not every such mutation will manifest phenotypically. In many triplets, A change in the third base has no consequences ("silent" mutations). Even a substitution of the first or second base of a triplet does not always lead to serious consequences. Although higher-order structures (Tertiary and Quaternary) are determined by the Primary Protein Structure (i.e., the Amino Acid Sequence), different Amino acids play roles of varying importance within that structure. For example, the mutation AUC → GUC leads to the replacement of isoleucine with valine, which is the substitution of one lipophilic group for another. However, the mutation CUU → CCU results in the replacement of leucine by Proline, and the consequence of such a substitution is a deviation from the normal spatial configuration of the polypeptide chain, which can drastically alter higher-order structure. It is thus evident that various mutations within the same structural gene of a given enzyme can affect its activity differently: any outcome is possible, ranging from a barely noticeable decrease in catalytic action to complete inactivation.

Back mutations and reversions. From the foregoing, it becomes clear that a mutant can undergo a back mutation that restores the wild-type properties. A true back mutation is spoken of only when a second mutation precisely restores the original genotype—that is, when the triplet altered by the first mutation once again codes for the exact same amino acid as before. If, however, the change merely results in the restoration of the original phenotype (e.g., the resumption of synthesis of a normally functioning enzyme), it is referred to as a reversion or suppressor mutation, and the respective cells are called revertants. Suppressor mutations can occur either within the original gene or at other sites within the chromosome (intragenic and extragenic suppressor mutations).

Induced Mutations

The mutation frequency can be increased by treating cells with mutagenic (mutation-causing) substances. In this case, one speaks of mutation induction, and the resulting cells are called induced mutants. Mutagens can be chemical, physical, or biological agents. The mechanisms of their action will be illustrated through several Examples.

Regarding their genetic structure, mutants are divided into three classes characterized by the following defects: (1) one base pair is replaced by another, such as AT replaced by GC or vice versa; (2) an additional base pair is inserted into The nucleotide sequence, or one of the existing pairs is lost; (3) a block of Base Pairs or even entire genes is lost (deletion), translocated within the chromosome (transposition), or "disrupted" by the insertion of foreign DNA (insertion).

Class 1 mutations, also referred to as point mutations, are characterized by a high frequency of reversion. In the case of class 2 mutations, which include frameshift mutations (see Fig. 15.4), revertants are rare, whereas following class 3 mutations (with a few exceptions), no revertants appear.

In the following sections, we will examine certain mechanisms of mutagenesis.

Incorporation of base analogs. Base analogs are antimetabolites. Some analogs closely resemble normal pyrimidine and purine bases that they are taken up by cells and incorporated into DNA. Once there, they largely function like normal bases, but unlike them, they exhibit a greater tendency to bind a "false" (inappropriate) partner during DNA replication. Bromouracil and 2-aminopurine are frequently used to induce mutations. Bromouracil is a compound structurally similar to thymine, which is incorporated into the DNA chain in place of thymine as a partner for adenine (Fig. 15.3). Bromouracil tautomerizes to the enol form more frequently than thymine. During the replication of a strand containing bromouracil, the analog in its enol form pairs with cytosine, thereby inducing the incorporation of guanine instead of adenine. Thus, in some cases, an AT base pair is replaced by a CG pair. 2-Aminopurine is incorporated into DNA in place of adenine and acts in a similar manner. This type of alteration—the substitution of one purine for another purine (A→G) or one pyrimidine for another pyrimidine (C → T)—is called a transition.

Fig. 15.3. Replacement of an AT base pair by a GC pair following the incorporation of bromouracil (BU) into DNA. A. Incorporation of several BU residues in place of thymine During the first replication (1-parental strand, 1'-daughter strand). B. Incorporation of G in place of A during the second replication as a result of pairing with BU in the enol form. C. Incorporation of C during the third replication.

Chemical modification of bases. Certain mutagenic substances act by chemically altering the bases contained within DNA, leading to replication errors. Nitrous acid causes a readily understandable alteration. Nitrous acid deaminates adenine, guanine, or cytosine without breaking or otherwise altering the polynucleotide chain. As a result of substituting an amino group with a hydroxyl group, adenine is converted into hypoxanthine, which pairs with cytosine instead of thymine, leading to an AT → GC mutation. If cytosine is deaminated to uracil, it pairs with adenine instead of guanine, leading to a GC → AT mutation. When converted into xanthine, guanine continues to pair with cytosine; hence, the deamination of G does not cause a mutation. Hydroxylamine reacts primarily with cytosine, modifying it such that it pairs with adenine; consequently, it also induces CG → TA mutations.

Alkylating agents. Ethyl- and methyl methanesulfonate, dimethyl and diethyl sulfate, ethyleneimine, nitrogen or sulfur mustard, and N-methyl-N'-nitro-N-nitrosoguanidine belong to the most potent mutagens. For instance, ethyl methanesulfonate predominantly alkylates the N atom of guanine. The resulting 7-alkylguanine is cleaved from the chain, leaving a "gap" in its place. During subsequent replication, an "erroneous" base often occupies this site.

Insertion or loss of individual base pairs. Proflavin and other acridine Dyes act differently. Presumably, an acridine molecule intercalates between adjacent bases of the DNA chain, increasing the distance between them (intercalation). Such spatial alteration during DNA replication can cause Two Types of errors: the loss of a nucleotide or the incorporation of an extra nucleotide pair. Mutations of this type lead to very serious consequences because they disrupt the reading frame during Protein Synthesis: starting from the site of nucleotide loss or insertion, the information is read in "incorrect" triplets (frameshift mutation, Fig. 15.4).

Fig. 15.4. Alteration in the reading frame of a nucleotide sequence as a result of a "frameshift mutation". Bacteriophage T4 is capable of producing Lysozyme. This enzyme is encoded by a phage gene. The top shows a segment of a normal nucleotide sequence (wild-type phage) with the corresponding amino acids indicated. The bottom shows the nucleotide sequence of a double mutant obtained from the wild type by two successive treatments with proflavin. Nucleotide A In the second triplet is lost, and from this point onward, the triplets are read incorrectly (the "reading frame" is shifted). The incorporation of G at the end of the fifth incorrect triplet subsequently restores the correct reading order. Thus, The nucleotide sequences of the double mutant and the wild type differ only in the region from the second to the fifth triplet inclusive. If the amino acids encoded by these triplets are not essential for the function of the given protein, the second mutation restores the wild-type properties (phenotype) (genetic suppression).

Ultraviolet rays and ionizing radiation. UV light, X-rays, and other Types of ionizing radiation exert both life-suppressing (lethal) and mutagenic effects on microorganisms. Their specific MECHANISM OF ACTION remains poorly understood. Based on the coincidence of the nucleic acid absorption curve and the cell survival inhibition curve upon irradiation as a function of wavelength, as well as the mutation frequency in the population, it can be concluded that UV rays act primarily on Nucleic Acids. Rays in the near-UV region with a wavelength of about 260 nm are the most effective (Fig. 15.5). Side damage in this case is negligible. Pyrimidine bases are predominantly affected. For example, two adjacent thymine bases in DNA can become covalently linked. The presence of such thymine dimers subsequently serves as a source of errors during replication (Fig. 15.6).

DNA Repair. Studies on UV-irradiated bacteria have demonstrated that DNA damage is partially reversible. If a bacterial suspension is irradiated with a high dose of ultraviolet light, a significant proportion of cells will suffer lethal damage (resulting in lethal mutations); if incubation in the dark is initiated immediately afterward, only a few cells will form colonies. However, if the cells are exposed to longer-wavelength light (320–550 nm) immediately after UV irradiation, the proportion of surviving cells increases several dozen times. This photoreactivation involves an enzyme activated by light that restores the normal Introduction/20.html">DNA Structure by splitting the formed thymine dimers. There is another repair mechanism that does not require light. In this dark reactivation, defective segments of the DNA chain are excised and replaced with new NUCLEOTIDES. The efficiency of such radiation damage repair varies among different bacterial strains. The radiation resistance characteristic of certain bacteria (e.g., Micrococcus radiodurans) is due to a highly efficient repair mechanism.

Fig. 15.5. Rationale for using a low-pressure mercury lamp to induce mutations and destroy microorganisms. The absorption spectrum of Nucleic Acids and the curve describing the bactericidal action of Light as a function of wavelength both peak at 260 nm. It is precisely in this region that the emission spectrum of a low-pressure mercury lamp exhibits a strong emission band (254 nm). Radiation from such a UV lamp exerts a sterilizing effect.

Fig. 15.6. Scheme of changes occurring in DNA upon UV irradiation and the processes of photoreactivation and dark reactivation. See text for explanations.

Transposon-induced mutations. In bacterial genetics, the method of obtaining mutations using Transposons is gaining increasing importance. Transposons (Tn) are short double-stranded DNA molecules consisting of more than 2,000 base pairs that typically confer resistance to a single antibiotic, or in exceptional cases, to several. Transposons are capable of "jumping" from one region of The Genome to another—specifically, from the bacterial chromosome to a plasmid and back—and can thus integrate into various genomic sites (see Section 15.3.1). If a transposon inserts into a structural gene of the chromosome, the nucleotide sequence of that gene is disrupted, and the Genetic information cannot be translated into a functionally complete polypeptide. An insertion mutant is thereby generated.

Since transposons are incapable of autonomous replication, their transfer from one bacterial cell to another requires a so-called vector (carrier). Plasmids or Bacteriophages can serve as vectors. It is worth noting that coliphage Mu (the "mutator phage"), much like a transposon, has The ability to insert into various sites of the bacterial chromosome and induce mutations. For this reason, phage Mu has been termed a "giant transposon" and is routinely used in the practical generation of E. coli mutants.

Expression of traits. The mere possibility of photoreactivation after UV irradiation indicates that the primary effect of a mutagenic factor does not necessarily lead to a true mutation. The incorporation of bromouracil into the DNA chain or the dimerization of thymine represents merely a premutation; thymine dimerization is a reversible process, and in the case of photoreactivation, it does not result in the appearance of a mutant. Only upon subsequent reduplication of the premutated DNA chain does the primary lesion become stable and subsequently pass on to progeny as a new element of the genotype. Such a fixed mutation can disappear only as a result of a reverse mutation. The phenotypic expression of a mutation involves a series of sequential processes that require a certain amount of time or several cell divisions. A new phenotype appears only when the altered gene begins to function. The stages necessary for the realization of a new phenotype vary for different cells and different Types of mutations.

Delayed expression of mutations. If a reversion occurs in a haploid cell, converting an auxotrophic mutant cell into a prototrophic one, such a reverse mutation is immediately expressed in the phenotype. The restoration of the ability to produce a specific enzyme can be readily recognized under appropriate conditions. The situation is different for mutations that conversely lead to an auxotrophic state, such as the loss of the ability to synthesize a specific amino acid. Such mutations can only be identified after a period encompassing several cell generations. This delayed expression is explained by the fact that although the mutation renders the Synthesis of the essential enzyme impossible, the enzyme synthesized previously continues to function for some time. The new trait will manifest only when sufficient "dilution" of this enzyme occurs as a result of cell divisions. Delayed phenotypic change must also be taken into account when screening for phage-resistant bacteria. If phage-sensitive bacteria acquire resistance as a result of a mutation leading to the loss of the ability to synthesize a specific receptor substance, this resistance will only become apparent after this substance has been sufficiently diluted through a series of cell divisions.

In multinucleated (cenocytic) cells, a distinction must also be made between mutations leading to the gain of a function and those leading to its loss. Many bacteria possess multiple nuclei (Chromosomes). Escherichia coli cells growing rapidly in nutrient-rich media contain an average of four chromosomes each. In a gain-of-function mutation, the mutant chromosome dominates: it immediately triggers the synthesis of the new enzyme, and the mutation is expressed in the phenotype without delay. However, if a loss-of-function mutation occurs in a multinucleated cell, it proves to be recessive. During repeated cell divisions, the nuclei are distributed among different daughter cells (nuclear segregation, Fig. 15.7). The defect can only manifest in a cell in which all nuclei contain the mutated gene. The progeny of such a cell constitutes a genetically pure clone. Thus, in the case of multinucleated cells, both nuclear segregation and phenotypic expression must be taken into account for loss-of-function mutations.

Fig. 15.7. Scheme of nuclear segregation during the Reproduction of a multinucleated bacterium. A genetically pure clone is formed only after the third division. Several more cell generations may be required for the phenotypic expression of the mutation (to achieve sufficient dilution of the product present in the original cell).

15.2.3 Mutant Selection

On standard solid media, only a few mutations can be directly detected by changes in pigmentation, altered colony morphology, or other traits. Some mutant traits are revealed through The addition of indicators or dyes. To identify mutants differing from parental cells in their reduced or elevated nutritional requirements, one must compare the growth of both on two different media. If, for instance, a mutant has lost the ability to synthesize leucine—a capability possessed by parental (wild-type) cells—it will grow only on media supplemented with this amino acid. We refer to such a mutant as leucine-auxotrophic, i.e., requiring leucine (leu^-), and also as leucine-defective, contrasting it with the prototrophic wild type (leu⁺). If both leu^- mutant cells and prototrophic wild-type cells are present simultaneously in a cell suspension, the two types can be distinguished by their growth on two different media. The method commonly used to detect such defective mutants is illustrated in Fig. 15.8.

Fig. 15.8. Identification and isolation of mutants with biochemical defects. Three Petri dishes show the results of an experiment to identify Alcaligenes eutrophus mutants that have lost the ability to grow on a medium containing fructose (Fruc^-). The nutrient medium in dishes A and B contains lactate, on which both wild-type and mutant cells grow equally well. In dish B, the medium contains only fructose; mutants do not grow on it. A mixture of both cell types obtained from an enrichment culture using a modified penicillin method was plated onto dish A (master plate). After individual colonies formed, they were transferred using a velvet stamp to dishes B and C [Note: translating literally as plates B and C based on context]. Cells incapable of utilizing fructose were identified by the fact that they grew only on dish B but not on dish C [Note: keeping context of the original text's letters]. The inability of these mutants to utilize fructose is caused by a defect in 2-keto-3-deoxy-6-phosphogluconate aldolase. Defective colonies are indicated by arrows. A similar method is used to identify and isolate amino acid-auxotrophic mutants. (Photo by I. Sammler.)

Fig. 15.9. The penicillin method used for the accumulation and isolation of auxotrophic mutants of Escherichia coli or other penicillin-sensitive bacteria.

As already mentioned, the mutation frequency for many traits is extremely low. For most metabolic physiological traits studied to date, it ranges from 10^-10 to 10^-5. At a frequency of 10^-8, one would need to screen 100 million cells or their progeny to identify new mutants. Although mutagens significantly increase mutation frequency, the labor required to detect them would still be very high. Therefore, population enrichment for mutants is performed prior to direct selection.

Obtaining an enrichment culture of mutants resistant to Antibiotics, poisons, or bacteriophages is relatively straightforward. Only mutants resistant to the respective agent survive on a supplemented medium, whereas wild-type cells perish.

Methods for the enrichment of auxotrophic mutants are based on a common principle: the cell suspension is subjected to conditions where the mutants to be isolated do not grow, while the growing prototrophic cells are eliminated or destroyed. Certain agents affect only growing cells, leaving non-growing, "resting" cells unharmed. After removing the antimicrobial agent and adding the necessary growth factors, the auxotrophic cells begin to grow.

Enrichment of mutants using penicillin. Penicillin is commonly used when isolating auxotrophic mutants of E. coli. It kills growing wild-type cells, whereas non-growing mutant cells survive this Treatment (Fig. 15.9).

Fig. 15.10. Normal colonies and minute ("pinpoint") colonies of a defective Alcaligenes eutrophus mutant, grown on an agar medium containing fructose (1%) and succinate (0.01%). (Photo by I. Sammler.)

Following the mutation-inducing treatment and several hours of growth, the bacterial suspension is incubated in a glucose-containing medium lacking a nitrogen source. This step allows the cells to consume any remaining soluble nitrogen compounds. After a few hours, penicillin and ammonium sulfate are added, and incubation is continued (this time for up to 24 hours). The prototrophic parental cells grow and are consequently killed by penicillin, whereas the auxotrophic mutants, which require a specific amino acid, do not grow and thus survive. The suspension is then freed from penicillin by washing or by adding penicillinase, and plated onto an agar medium containing amino acids. The proportion of auxotrophic cells among the resulting colonies is significantly higher (alongside prototrophic cells that managed to survive the penicillin treatment). If the bacteria are penicillin-resistant, alternative antibiotics can be used for the same purpose (novobiocin, cycloserine, colistin, kanamycin). The selective destruction of growing cells can also be achieved through a phenomenon known as "Lethal synthesis".

These techniques allow for the enrichment and isolation of mutants with various defects, including impaired transport systems or substrate utilization, Intermediary Metabolism defects, and heightened Temperature sensitivity ("conditionally lethal" mutants). Later, we will discuss techniques for isolating other mutants with altered Metabolic Regulation. Table 15.2 summarizes the selection and identification methods for various mutant types, including regulatory mutants.

Table 15.2. Methods for the selection and identification of Different types of mutants