I.

INTRODUCTION
The mechanism for DNA replication in procaryotes is good example of the exchange of genetic
information from one generation to another. However some individuals can receive additional genetic
information through natural or artificial means. The initial genetic information within a cell may also
undergo rearrangements or alterations. This chapter will discuss some mechanisms causing alterations in
a cell’s content of genetic information and ways that we can manipulate those mechanisms to improve
bioprocesses.
1.1 Evolving Desirable Biochemical Activities through Mutation and Selection
Although the cell has a well-developed system to prevent errors in DNA replication and an active repair
system to correct damage to a DNA molecule, mistakes occur. These mistakes are called mutations.
Genotype – the sum of the genetic construction of an organism.
Phenotype – characteristics expressed by a cell constitute. The phenotypic response of a culture may
change reversibly with alterations in environmental conditions, whereas the genotype is constant
irrespective of the environment.
A mutation is a genotypic change and is irreversible. A whole culture undergoes a phenotypic response,
whereas only a rare individual will undergo a genotypic change. For example, if a culture changes in
color from white to green to white upon an increase in dissolved oxygen, the change would be
phenotypic. Now consider an alternative experiment where white cells are removed from a culture and
placed on a plate (small circular dish filled with nutrients solidified with agar) and allowed to grow into
separate colonies. If one colony remain green when cultures under the original conditions, it would be
evidence for genotypic change. In this case, the white cells would be the wild type and the green cells the
mutants.
1.2 How Mutations Occur
Most mutations occur due to mistakes in DNA synthesis. Some examples are shown in Fig. 1.1. One
common form is a point mutation. A point mutation results from the change of a single base (for
example, cytosine instead of thymine). Some point mutations are silent mutations because the altered
codon still codes for the same amino acid (e.g., UCU and UCA both code for serine). Even if the point
mutation causes the substitution of different amino acids, it may or may not alter protein activity greatly,
whereas the same substitution at another site might not be very critical.
One type of point mutation that usually has a profound effect results in a nonsense or stop codon (e.g.,
CAA for glutamine to UAA for stop on the m-RNA from the altered DNA). This results in the premature
termination of translation and an incompletely formed protein.
Generally, deletion mutations have profound effects on cellular metabolism. By deleting or adding one or
more bases, we can alter the whole composition of a protein, not just a single amino acid. A deletion can
shift the reading frame when translating the resulting m-RNA. This illustrated in Fig 1.2.
Additions often take place through insertion elements (IS). These elements are about 700 to 1400 base
pairs in length; in E.coli about five different IS sequences are known and are present on the chromosome.
These elements can move on the chromosome from essentially any one site to another. Often they will
insert in the middle if a gene, totally destroying its function.
Back mutations or reversions are possible. Revertants are cells for which the original wild type
phenotype has been restored. Restoration of a function can occur due to a change at the original mutation
(e.g., if the original mutation was CAA to UAA, then a second mutation for UAA to CAA restores the
original genotype and phenotype). Second-site revertants can occur that restore phenotype (suppressor
mutations), but not genotype (e.g., a second deletion mutation that restores the gene to the normal reading
frame or a mutation is another gene that restores the wild-type phenotype).

Figure 1.1. DNA base changes from wild type, involving point mutation and deletion.

1.1.2 Selecting for Desirable Mutants

Mutants can serve as powerful tools to better understand cell physiology; they are also
valuables as industrial organism, because mutation can be used to alter metabolic regulation and
to cause overproduction of a desired compound. Methods to induce mutations and then select for
mutants are important tools for catalyst development in bioprocessing.

Natural (spontaneous rates of mutation vary greatly from gene to gene (10−3 𝑡𝑜 10−9 per
cell division), with 10−6 mutations in a gene per cell division being typical. Chemical agents
(mutagens) or radiation are often used in the laboratory to increase mutation rates. Mutagens are
nonspecific and may affect any gene.
 Figure 1.2.Effect of deletion of a base on the reading frame and the protein encoded.
The selection of a mutant with desirable properties is no easy task. Mutations are classified as selectable
and unselectable. A selectable mutation confers upon the mutant and advantage for growth or survival
under specific set of environmental conditions; thus, the mutant can grow and the wild type will die. An
unselectable mutant requires a cell-by-cell examination to find a mutant with the desired characteristics
(e.g., green pigment). Even the mutagens, the frequency of mutation is sufficiently low to make
prohibitive a brute-force screening effort for most unselectable mutants.
Selection can be direct or indirect. An example of direct selection would be to find a mutant resistant to
an antibiotic or toxic compound. A culture fluid containing 108 𝑡𝑜 1010 cells/ml is subjected to a
mutagenic agent. A few drops of culture fluid are spread evenly on a plate, with the antibiotic
incorporated into the gelled medium. Only antibiotic resistant cells can grow, so any colonies that form
must arise from antibiotic-resistant mutants. If one in a million cells has this particular mutation, we
would expect to find about 10 to 100 colonies per plate if 0.1 ml of culture fluid was tested.
Indirect selection is used for isolating mutants that are deficient in their capacity to produce a necessary
growth factor (e.g., an amino acid or a vitamin). Wild-type E.coli grows on glucose and minerals salts.
Auxotrophic mutants would not grow on such a simple medium unless it were supplemented with the
growth factor that the cell could no longer make (e.g., a lysine auxotroph has lost the capacity to make
lysine, so lysine must be added to the glucose and salts to enable to cell to grow). The wild-type cell that
needs no supplements to a minimal medium is a prototroph. Consider the selection of a rare mutant cell
that is auxotrophic for lysine from a population of wild-type cells. This cannot be done directly, since
both cell types would grow in the minimal medium supplemented with lysine. A method that facilitates
selection greatly is called replicate plating (see fig 8.3). a master plate using lysine-supplemented
medium will grow both the auxotroph and wild-type cells. Once colonies are well formed on the master
plate, an imprint is made on sterile velveteen. The bristles on the velveteen capture some of the cells
from each colony. The orientation of the master plate is carefully noted. Then a test plate with minimal
medium is pressed against the velveteen: some cells, at the point of each previous colony, then serve to
inoculate the test plate at positions identical to those on the master plate. After incubation (approximately
24 hours for E.coli), the test plate is compared to the master plate. Colonies that appear at the same
positions on both plates arise from wild-type cells, while colonies that exist only on the master plate must
arise from the auxotrophic mutants.
Another class of mutants is called conditional mutants. Mutations that would normally be lethal to the
cell could not be detected by methods we have described so far. However, mutated proteins are often
more temperature sensitive than normal proteins. Thus, temperature sensitivity can often be used to select
for conditionally lethal mutations. For example, the mutant may be unable to grow at the normal growth
temperature (e.g., 37˚C for E.coli) but will grow satisfactory at a lower temperature (e.g., 25˚C). Table
8.1 summarizes a variety of mutants and how they may be detected.
Mutation and selection have been used to tremendous advantage to probe and basic features of cell
physiology and regulation. They also have been the mainstay of industrial programs for the improvement
of production strains. Mutation and selection programs have been primarily responsible for increasing the
yield of penicillin from 0.001 g/l in1939 to current value of about 50 g/l of fermentation broth.

Figure 1.3 Replica-plating methods for detecting nutritional mutants.

Table 1.1 Kinds of Mutants

Description Nature of Change Detection of Mutant:

Nonmotile Loss of flagella; non-functional flagella Compact colonies instead of flat,
spreading colonies
Noncapsulated Loss or modification surface capsule Small, rough colonies instead of
larger, smooth colonies
Rough colony Loss or change in lipopolysaccharide outer Granular, irregular colonies instead of
layer smooth, glistening colonies
Nutritional Loss of enzyme in biosynthetic pathway
Sugar fermentation Loss of enzyme in degradative pathway
Drug resistant Impermeability to drug or drug target is
altered or drug is detoxified
Virus resistant
Temperature sensitive
Cold sensitive

SOURCE:
http://utminers.utep.edu/rwebb/html/isolation_of_spontaneous_mutat.html