Microbiology and Immunology

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Mutations

405

•

Cells detect external alkalization with the help of a

mechanism known as the alkaline signal transductionsystem.

Under such environmental conditions, an inducible system for

internal pH homeostasis works in E. coli.The so-called

sodium-proton antiporter gene NhaA is induced at high exter-

nal pH in the presence of high sodium. The NhaA antiporter

acts to acidify the cytoplasm through proton/sodium

exchange. This allows the microorganism to survive above its

normal pH range. As B. alkalophilusmay have as many as

three sodium-proton antiporters, it is felt that the number of

antiporters may relate to the alkalophilicity of a species.

The search for extremophileshas intensified recently.

Standard enzymesstop working when exposed to heat or other

extreme conditions, so manufacturers that rely on them must

often take special steps to protect (stabilize) the proteins dur-

ing reactions or storage. By remaining active when other

enzymes would fail, enzymes from extremophiles

(extremozymes) can potentially eliminate the need for those

added steps, thereby increasing efficiency and reducing costs

in many applications.

Many routes are being followed to use the capacity that

such extremophiles possess. First, the direct use of these natu-

ral mutants to grow and produce the useful products. Also, it

is possible with recombinant DNA technology to isolate genes

from such organisms that grow under unusual conditions and

clone them on to a fast growing organism. For example, an

enzyme alpha-amylase is required to function at high temper-

ature for the hydrolysis of starch to glucose. The gene for the

enzyme was isolated from Bacillus stearothermophilus,an

organism that is grows naturally at 194°F (90°C), and cloned

into another suitable organism. Finally, attempts are being

made to stabilize the proteins themselves by adding some

groups (e.g., disulfide bonds) that prevent its easy denatura-

tion. This process is called protein engineering.

Conventional mutagenesis and selectionschemes can

be used in an attempt to create and perpetuate a mutant form

of a gene that encodes a protein with the desired properties.

However, the number of mutant proteins that are possible after

alteration of individual nucleotides within a structural gene by

this method is extremely large. This type of mutagenesis also

could lead to significant decrease in the activity of the

enzyme. By using set techniques that specifically change

amino-acids encoded by a cloned gene, proteins with proper-

ties that are better than those obtained from the naturally

occurring strain can be obtained. Unfortunately, it is not pos-

sible to know in advance which particular amino acid or short

sequence of amino acids will contribute to particular changes

in physical, chemical, or kinetic properties. A particular prop-

erty of a protein, for example, will be influenced by amino

acids quite far apart in the linear chain as a consequence of the

folding of the protein, which may bring them into close prox-

imity. The amino acid sequences that would bring about

change in physical properties of the protein can be obtained

after characterization of the three dimensional structure of

purified and crystallized protein using x-ray crystallography

and other analytical procedures. Many approaches are being

tried to bring about this type of “directed mutagenesis” once

the specific nucleotide that needs to be altered is known.

See alsoBacterial adaptation; Evolutionary origin of bacteria

and viruses; Microbial genetics; Mutations and mutagenesis

MMutationsUTATIONS

A mutation is any change in genetic material that is passed on

to the next generation. The process of acquiring change in

genetic material forms the fundamental underpinning of evo-

lution. Mutation is a source of genetic variation in all life

forms. Depending on the organism or the source of the muta-

tion, the genetic alteration may be an alteration in the organ-

ized collection of genetic material, or a change in the

composition of an individual gene.

Mutations may have little impact, or they may produce a

significant positive or negative impact, on the health, competi-

tiveness, or function of an individual, family, or population.

Mutations arise in different ways. An alteration in the

sequence, but not in the number of nucleotides in a gene is a

nucleotide substitution. Two types of nucleotide substitution

mutations are missense and nonsense mutations. Missense

mutations are single base changes that result in the substitu-

tion of one amino acid for another in the protein product of the

gene. Nonsense mutations are also single base changes, but

create a termination codon that stops the transcriptionof the

gene. The result is a shortened, dysfunctional protein product.

Another mutation involves the alteration in the number

of bases in a gene. This is an insertion or deletion mutation.

The impact of an insertion or deletion is a frameshift, in which

the normal sequence with which the genetic material is inter-

preted is altered. The alteration causes the gene to code for a

different sequence of amino acids in the protein product than

would normally be produced. The result is a protein that func-

tions differently—or not all—as compared to the normally

encoded version.

Genomes naturally contain areas in which a nucleotide

repeats in a triplet. Trinucleotide repeat mutations, an

increased number of triplets, are now known to be the cause of

at least eight genetic disorders affecting the nervous or neuro-

muscular systems.

Mutations arise from a number of processes collectively

termed mutagenesis. Frameshift mutations, specifically inser-

tions, result from mutagenic events where DNAis inserted into

the normally functioning gene. The genetic technique of inser-

tional mutagenesis relies upon this behavior to locate target

genes, to study gene expression, and to study protein struc-

ture-function relationships.

DNA mutagenesis also occurs because of breakage or

base modification due to the application of radiation, chemicals,

ultraviolet light, and random replication errors. Such mutagenic

events occur frequently, and the cell has evolved repair mecha-

nisms to deal with them. High exposure to DNA damaging

agents, however, can overwhelm the repair machinery.

Genetic research relies upon the ability to induce muta-

tions in the lab. Using purified DNA of a known restriction

map, site-specific mutagenesis can be performed in a number

of ways. Some restriction enzymesproduce staggered nicks at

the site of action in the target DNA. Short pieces of DNA

womi_M 5/7/03 7:53 AM Page 405