As precise and predictable as the rules of genetic expression seem, permanent changes do occur in the genetic code. Indeed, genetic change is the driving force of evolution through its addition of variation to populations of organisms. Changes may become evident in altered gene expression, such as in the appearance or disappearance of anatomical or physiological traits. For example, a pigmented bacterium can lose its ability to form pigment, or a strain of the malarial parasite can develop resistance to a drug. A phenotypic change that is due to an alteration in the genotype is called a mutation. On a molecular level, a mutation is an alteration in the base sequence of DNA. It can involve the loss of base pairs, the addition of base pairs, or a rearrangement in the order of base pairs. Do not confuse this with genetic recombination, in which microbes transfer whole segments of genetic information between themselves.
A microorganism that exhibits a natural, nonmutated characteristic is known as a wild type or wild strain. If a microorganism develops a mutation, it is called a mutant strain. Mutant strains can show variance in morphology, nutritional characteristics, genetic mechanisms, resistance to chemicals, temperature preference, and nearly any type of enzymatic function. Mutant strains are useful for tracking genetic events, unraveling genetic organization, and pinpointing genetic markers.
The simplest way to detect mutant bacteria is to inoculate solid media containing differential or selective agents such as metabolic substrates or antibiotics. Growing a pure culture on a plate of media and observing the appearance of colonies can reveal strains of a species that express genetic variations. For instance, in cultures of wild-type E. coli grown on differential MacConkey agar, most of the isolated colonies will indicate a positive result for lactose fer mentation, but invariably there will be a small number of colonies that are negative and have lost the genes for using lactose. This colony can be readily isolated for further study. Antibiotics are frequently added to media as selective agents for isolating strains of bacteria that are drug resistant.
Causes of Mutations
A mutation is described as spontaneous or induced, depending upon its origin. A spontaneous mutation is a change in the DNA arising from errors in replication that occur without a known cause. The frequency of spontaneous mutations can range from 1 mutation every 10,000 bases to 1 per 10,000,000,000, oftentimes related to the complexity of the DNA at that point. Areas of the DNA that are repetitive or show a great deal of secondary structure such as loops or hairpins (figure 1) tend to have higher mutation rates.
Fig1. Secondary structure in DNA can lead to increased mutation rates. (a) Stretches of DNA that are repetitive may lead to mutations as the DNA polymerase skips a nucleotide or reads the same nucleotide twice. (b) Sequences that allow complementary base pairing within the DNA strand may cause secondary structures (loops, bends, or hairpins) to form in the DNA, which also increases the mutation rate.
Induced mutations result from exposure to known mutagens, which are physical or chemical agents that damage DNA and inter fere with its function (table 1). The carefully controlled use of mutagens has proved a useful way to induce mutant strains of microorganisms for study.
Table1. Selected Mutagenic Agents and Their Effects
Chemical mutagenic agents act in a variety of ways to change the DNA. Agents such as acridine dyes insert completely across the DNA helices between adjacent bases. This results in distortion of the helix and can cause frameshift mutations. Analogs5 of the bases (5-bromodeoxyuridine and 2-aminopurine, for example) are chemical mimics of natural bases that are incorporated into DNA during replication. The addition of these abnormal bases leads to mistakes in base-pairing. Many chemical mutagens are also carcinogens, or cancer-causing agents.
Physical agents that alter DNA are primarily types of radiation. High-energy gamma rays and X-rays introduce major physical changes into DNA, which can accumulate breaks that may not be repairable. Ultraviolet (UV) radiation induces abnormal bonds between adjacent pyrimidines so that normal replication of that region is blocked. Exposure to large doses of radiation can be fatal, which is why radiation is so effective in microbial control; it can also be carcinogenic in animals.
Categories of Mutations
Mutations range from large mutations, in which long genetic sequences are gained or lost, to small ones that affect only a few bases on a gene. These latter mutations, which involve addition, deletion, or substitution of no more than a few bases, are called point mutations.
To understand how a change in DNA influences the cell, re member that the DNA code appears in a particular order of triplets (three bases) that is transcribed into mRNA codons, each of which specifies an amino acid. A permanent alteration in the DNA that is copied faithfully into mRNA and translated can change the structure of the protein. A change in a protein can likewise change the morphology and physiology of a cell. Most mutations have a harmful effect on the cell, leading to cell dysfunction or death. Neutral mutations produce neither adverse nor helpful changes. A small number of mutations are beneficial in that they provide the cell with a useful change in structure or physiology.
Any change in the code that leads to incorporation of a different amino acid in the protein is called a missense mutation. A missense mutation can do one of the following:
● create a faulty, nonfunctional (or less functional) protein,
● produce a protein that functions in a different manner, or
● cause no significant alteration in protein function.
A nonsense mutation, on the other hand, changes a normal co don into a stop codon that does not code for an amino acid and stops the production of the protein wherever it occurs. A nonsense mutation almost always results in a nonfunctional protein. A silent mutation alters a base but does not change the amino acid and thus has no effect. For example, because of the redundancy of the code, ACU, ACC, ACG, and ACA all code for threonine, so a mutation that changes only the last base will not alter the sense of the message in any way. A back-mutation occurs when a gene that has undergone mutation reverses (mutates back) to its original base composition.
Mutations also occur when one or more bases are inserted into or deleted from a newly synthesized DNA strand (see table 2, IIB). This type of mutation, known as a frameshift, is so named because the reading frame of mRNA will shift to the left or right, depending on the type of change. Frameshift mutations nearly always result in a nonfunctional protein because the codon structure will become reset from that point on, will code for a different sequence of amino acids from the original DNA, and is likely to introduce stop codons. Also note that insertion or deletion of bases in multiples of three (3, 6, 9, etc.) does not disturb the reading frame downstream from the mutation site. It can still disrupt the structure of the protein, depending on the change in the amino acid sequence. The effects of these types of mutations is modeled in table 2.
Table2. Classification of Major Types of Mutations
Repair of Mutations
Earlier, we indicated that DNA has a proofreading mechanism to repair mistakes in replication that might otherwise become permanent. Because mutations are potentially life-threatening, the cell has additional systems for finding and repairing DNA that has been damaged by various mutagenic agents and processes. Most ordinary DNA damage is resolved by enzymatic systems specialized for finding and fixing such defects.
DNA that has been damaged by ultraviolet radiation can be restored by photoactivation or light repair. This repair mechanism requires visible light and a light-sensitive enzyme, DNA photolyase, which can attach to sites of abnormal pyrimidine bonding and restore the original DNA structure. Ultraviolet repair mechanisms are successful only for a relatively small number of UV mutations. Mutations can be excised by a series of enzymes that remove the incorrect bases and add the correct ones. This process is known as excision repair. First, enzymes break the bonds between the bases and the sugar-phosphate strand at the site of the error. A different enzyme subsequently removes the defective bases one at a time, leaving a gap that will be filled in by DNA polymerase I and ligase. A repair system can also locate mismatched bases that were missed during proofreading: for example, C mistakenly paired with A, or G with T. The base must be replaced soon after the mismatch is made, or it will not be recognized by the repair enzymes.
The Ames Test
New agricultural, industrial, and medicinal chemicals are constantly being added to the environment, and exposure to them is widespread. The discovery that many such compounds are muta genic and that nearly all mutagens are linked to cancer is significant. Although animal testing has been a standard method of detecting chemicals with carcinogenic potential, a more rapid screening system called the Ames test6 is also commonly used. In this inventive test, the experimental subjects are bacteria whose gene expression and mutation rate can be readily observed and monitored. The premise is that any chemical capable of mutating bacterial DNA can similarly mutate mammalian (and thus human) DNA and is therefore potentially hazardous. Many potential carcinogens (benzanthracene and aflatoxin, for example) are muta genic agents only after being acted on by mammalian liver enzymes, so an extract of these enzymes is added to the test medium.
One indicator organism in the Ames test is a mutant strain of Salmonella enterica that has lost the ability to synthesize the amino acid histidine, a defect highly susceptible to back-mutation because the strain also lacks DNA repair mechanisms. Mutations that cause reversion to the wild strain, which is capable of synthesizing histidine, occur spontaneously at a low rate. A test agent is considered a mutagen if it enhances the rate of back-mutation beyond levels that would occur spontaneously. Figure 2 compares a standard version of the test with an automated method that streamlines result taking. The Ames test has proved invaluable for screening an assortment of environmental and dietary chemicals for mutagenicity and carcinogenicity without resorting to animal studies.
Fig2. The Ames test. (a, b, c) In this traditional plate method, a strain of Salmonella enterica that cannot synthesize histidine [his(−)] is the test species. If the chemical causes an increase in mutation rate to histidine-producing cells as compared to a control, the chemical is considered mutagenic. (d) The newer well fluctuation test is based on the same basic scheme; however, it has greater sensitivity and is easier to evaluate. (d): Xenometrix AG
Positive and Negative Effects of Mutations
Many mutations are not repaired. How the cell copes with them depends on the nature of the mutation and the strategies available to that organism. Mutations are passed on to the offspring of organ isms during reproduction and to new viruses during replication. They become a long-term part of the gene pool. Most mutations are harmful to organisms, but some can provide adaptive advantages.
If a mutation leading to a nonfunctional protein occurs in a gene for which there is only a single copy, as in haploid organisms, the cell will probably die. This happens when certain mutant strains of E. coli acquire mutations in the genes needed to repair damage by UV radiation. Mutations of the human genome affecting the action of a single protein (mostly enzymes) are responsible for more than 3,500 diseases.
Although most spontaneous mutations are not beneficial, a small number contribute to the success of the individual and the population by creating variant strains with alternative ways of ex pressing a trait. Microbes are not “aware” of this advantage and do not direct these changes; they simply respond to the environment they encounter. In the long-range view, mutations and the variations they produce are the raw materials for change in the population and, thus, for evolution.
Mutations that create variants occur frequently enough that any population contains mutant strains for a number of characteristics, but as long as the environment is stable, these mutants will never comprise more than a tiny percentage of the population. When the environment changes, however, it can become hostile for the survival of certain individuals, and only those microbes bearing protective mutations will be equipped to survive in the new environment.
Such observations can be explained by the important biological principles of natural selection and survival of the fittest. This theory states that environmental pressure selects for organisms that have greater adaptability due to beneficial mutations. They are more likely to survive, reproduce, and pass the changed genes on to succeeding generations. In time, these survivors become the dominant members of the population as long as the mutation continues to favor their survival. One of the clearest models for natural selection in action is acquired drug resistance in bacteria.
Bacteria have additional mechanisms for changing their genetic makeup by sharing genes with other bacteria through recombination.
