Bacterial Mutations

Mutation: Introduction

All organisms contain a specific sequence of nucleotide bases in their genome, their genetic blueprint. A mutation is a heritable change in the base sequence of the nucleic acid in the genome of an organism or a virus or any other genetic entity. In all cells, the genome consists of double stranded DNA. In viruses, by contrast, the genome may consist of single- or double-stranded DNA or RNA.

A strain of any cell or virus carrying a change in nucleotide sequence is called a mutant. A mutant by definition differs from its parental strain in its genotype, the nucleotide sequence of the genome. In addition, the observable properties of the mutant—its phenotype— may also be altered relative to its parent. This altered phenotype is called a mutant phenotype. It is common to refer to a strain isolated from nature as a wild-type strain. The term wild-type may be used to refer to a whole organism or just to the status of a particular gene that is under investigation. Mutant derivatives can be obtained either directly from wild-type strains or from other mutants.

Genotype and Phenotype

Depending on the mutation, a mutant strain may or may not differ in phenotype from its parent. By convention in bacterial genetics, the genotype of an organism is designated by three lowercase letters followed by a capital letter (all in italics) indicating a particular gene. For example, the hisC gene of Escherichia coli encodes a protein called HisC that functions in biosynthesis of the amino acid histidine. Mutations in the hisC gene would be designated as hisC1, hisC2, and so on, the numbers referring to the order of isolation of the mutant strains.

Wild and mutant bacterial colonies

Spontaneous Mutation and Induced Mutation

Mutations can be either spontaneous or induced. Induced mutations are those that are due to agents in the environment and include mutations made deliberately by humans. They can result from exposure to natural radiation (cosmic rays, and so on) that alters the structure of bases in the DNA. In addition, a variety of chemicals, including oxygen radicals, can chemically modify DNA. For example, oxygen radicals can convert guanine into 8-hydroxyguanine, and this causes mutations. Spontaneous mutations are those that occur without external intervention. The bulk of spontaneous mutationsresult from occasional errors in the pairing of bases during DNA replication.

Point mutation and Chromosomal aberrations

In eukaryotes, the mutation is in large scale at part of chromosomal level, (addition / deletion / duplication / translocation etc), and referred as Chromosomal aberrations. Whereas in bacteria like prokaryotic organisms, the mutation changes one or two basepairs, which are referred as Point mutations.

If a point mutation is within the coding region of a gene that encodes a polypeptide, any change in the phenotype of the cell is most likely the result of a change in the amino acid sequence of the polypeptide. The error in the DNA is transcribed into mRNA, and the erroneous mRNA in turn is translated to yield apolypeptide.

Base pair substitutions and frame-shift mutations are the two point mutations common in bacteria.

Chromosomal aberrations

Point mutation

Base Pair substitution

Base pair substitution refers replacement of one nucleotide by another and thus causing change in the amino acid sequence of the protein.

A purine is replaced by another purine or a pyrimidine is replaced by another pyrimidine is called as transition. A purine is replaced by pyrimidine or vice versa is referred as transversion.

Due to base pair substitution, a change in the mRNA during transcription resulted changes in the aminoacid compositions of the protein. Accordingly the effects of base pair substitutions can be of three types.

Silent mutation in which mutation that does not affect the phenotype of the cell. Even though a change in the base pair of the nucleotide it does not affect the composition of aminoacids (Four or two triplet codons are available for a single aminoacid, hence the change also gives the same aminoaicd).

Changes in the first or second base of the codon more often lead to significant changes in the polypeptide. For instance, a single base change from UAC to AAC results in an amino acid change within the polypeptide from tyrosine toasparagine at a specific site. This is referred to as a mis sense mutation because the informational “sense” (precise sequence of amino acids) in the ensuing polypeptide has changed.

Another possible outcome of a base-pair substitution is the formation of a nonsense (stop) codon. This results in premature termination of translation, leading to an incomplete polypeptide that would almost certainly not be functional. Mutations of this type are called nonsense mutations because the change is from a codon for an amino acid (sense codon) to a nonsense codon. Unless the nonsense mutation isvery near the end of the gene, the incomplete product will be completely inactive.

Base pair substitution and its effects

Base pair substitution causes three different effects

Frame-shift mutation

The genetic code is read from one end of the nucleic acid in consecutive blocks of three bases (that is, as codons), and hence any deletion or insertion of a single base pair results in a shift in the reading frame during translation. This type of mutation is referred as frame-shift mutation.

Single base insertions or deletions change the primary sequence of the encoded polypeptide, typically in a major way. Such micro-insertions or micro-deletions can result from replication errors. Insertion or deletion of two base pairs also causes a frame shift; however, insertion or deletion of three base pairs adds or removes a whole codon. This results in addition or deletion of a single amino acid in the polypeptide sequence. Although this may well be deleterious to protein function, it is usually not as bad as a frame-shift, which scrambles the entire polypeptide sequence after the mutation point.

Insertions or deletions can also result in the gain or loss of hundreds or even thousands of base pairs. Such changes inevitably result in complete loss of gene function. Some deletions are so large that they may include several genes. If any of the deleted genes are essential, the mutation will be lethal. Such deletions cannot be restored through further mutations, but only through genetic recombination. Larger insertions and deletions may arise as a result of errors during genetic recombination. In addition, many insertion mutations are due to the insertion of specific identifiable DNA sequences 700–1400 base pairs (bp) in length called insertion sequences, a type of transposable element.

Random mutagenesis and Site-Directed Mutagenesis

The mutations that we have considered thus far have been random,that is, not directed at any particular gene, hence referred as random mutagenesis. However, recombinant DNA technology and the use of synthetic DNA make it possible to induce specific mutations in specific genes.The approach of generating mutations at specific sites is called site-directed mutagenesis.

Reversion

Point mutations are typically reversible, a process known as reversion. Reversion an alteration in DNA that reverses the effects of a prior mutation. A revertant is a strain in which the original phenotype that was changed in the mutant is restored. Revertants can be of two types. In same-site revertants and second-site revertants.

If the mutation that restores activity is at the same site as the original mutation, then the revertant is referred as same-site revertant. On the other-hand, the mutation is at a different site in the DNA and can restore a wild-type phenotype, it is said to be second-site revertants.

The possible second-site mutations are

(1) a mutation somewhere else in the same gene that restores enzyme function, such as a second frame-shift mutation near the first that restores the original reading frame.

(2) a mutation in another gene that restores the function of the original mutated gene.

(3) A mutation in another gene that results in the production of an enzyme that can replace the mutated one.

The second site mutation is also known as suppression mutation, as it suppress the effects of first mutation.

How to select a mutant

The spontaneous mutation resulted a mutation frequency of 10^-8 and induced with about 10^-6, it is always difficult to identify the mutants from the wild types.

Virtually any characteristic of an organism can be changed by mutation. However, some mutations are selectable, conferring some type of advantage on organisms possessing them, whereas others are non-selectable, even though they may lead to a very clear change in the phenotype of an organism. A selectable mutation confers a clear advantage on the mutant strain under certain environmental conditions, so the progeny of the mutant cell are able to outgrow and replace the parent. A good example of a selectable mutation is drug resistance: An antibiotic-resistant mutant can grow in the presence of antibiotic concentrations that inhibit or kill the parent and is thus selected for under these conditions. It is relatively easy to detect and isolate selectable mutants by choosing the appropriate environmental conditions.

Selection is therefore an extremely powerful genetic tool, allowing the isolation of a single mutant from a population containing millions or even billions of parental organisms.

An example of a non-selectable mutation is color loss in a pigmented organism. Non-pigmented cells usually have neither an advantage nor a disadvantage over the pigmented parent cells when grown on agar plates, although pigmented organisms may have a selective advantage in nature. We candetect such mutations only by examining large numbers of colonies and looking for the “different” ones, a process called screening. Hence, screening refers the procedure that permit the sorting of organism by phenotype or genotype.

Replica Plating Technique

Although screening is more tedious than selection, methods are available for screening large numbers of colonies for certain types of mutations. For instance, nutritionally defective mutants can be detected by the technique of replica plating. A mutant with a nutritional requirement for growth is called an auxotroph, and the parent from which it was derived is called a prototroph. (A prototroph may or may not be the wild type. An auxotroph may be derived from the wild type or from a mutant derivative of the wild type.) For instance, mutants of E. coli with a His^- phenotype are histidine auxotrophs. Although of great utility, replica plating is nevertheless a screening process and it can be laborious to isolate mutants by screening.

In this technique, an imprint of colonies from a master plate is made onto an agar plate lacking the nutrient by using sterile velveteen cloth or filter paper. Parental colonies will grow normally, whereas those of the mutant will not. Thus, the inability of a colony to grow on medium lacking the nutrient signals that it is a mutant. The colony on the master plate corresponding to the vacant spot on the replica plate can then be picked, purified, and characterized.

Watch this video "How replica plating is used for bacterial gene mapping" [Youtube]

Detailed lecture on mutation types and causes (Youtube)

MUTAGENS

The chemical, physical and biological agents that can induce the mutation are referred as mutagens.

1. Chemical mutagens

The chemicals can be divided into four groups based on their action namely,

a. Base analogs are the chemicals that are incorporated in the DNA instead of a specific base pair and subsequently cause mutation.

Example: 5-bromouracil (Incorporated instead of T which ultimately cause replacement of AT to GC)

Example: 2-aminopurine (Incorporated instead of A which ultimately cause replacement of AT to GC)

b. Chemicals react with DNA, especially the nitrogen base of the nucleotide and causing mutation

Example: Nitrous oxide deaminates (removal of amine) A and C thereby causes mutation as AT to GC and GC to AT.

Example: Hydroxylamine reacts with C and causes mutation as GC to AT

c. Alkylating agents are reacting with N-base through alkylation (Addition of R group)

Ethyl methane sulfonate (EMS) Add methyl group on G lead to faulty pairing with T (GC to AT)

Nitrosoguanidine: Cross link the DNA strands and causes deletion mutation

d. Intercalating agents: These chemically structurally look-like basepairs and will interact with DNA sequence and causes mutation.

Acrydine orange and ethydium bromide: Insert between base pair and causes addition mutation

2. Physical agents

Both non-ionizing and ionizing radiations able to cause mutation. Non-ionizing radiation has wider use in the microbial mutations.

UV light – This non-ionizing radiation can be absorbed by purine and pyrimidine strongly at 260 nm. Two adjacent pyrimidines form a dimer, by which covalently joint. During replication, the DNA polymerase wrongly recognize the pyrimidine and add a wrong base pair which leads to mutation.

Gamma rays – This ionizing radiation at low concentrations react as free radical attack and DNA breakage which leads to mutation. Gamma rays are not commonly used for microbial mutations, instead, highly useful for mutating plant materials.

3. Biological agents

Transposable element, insertion sequences and Mu phage are some of the biological agents cause mutation.

Transposable element – A genetic element that can move from one place of chromosomal DNA to another place. Such element’s insertion lead to mutation which is referred as transposon mutagenesis
Insertion sequences – The simplest type of transposable sequence (about 700 – 1000 bp) which has the transposase gene alone, which causes the mutation.
Mu phage – This double strand DNA containing temperate bacteriophage can able to act as transposable elements, causes mutation by transposition.

Site directed Mutation:

So for we have discussed about the mutations which occur at random, that means, the gene which is mutated and type of mutation cannot be predicted, referred as random mutation. The present modern molecular techniques allow to mutate an organism at specific portion of gene by means of special vectors (plasmids) with controlled system. This type of mutation is referred as site directed mutation.

Watch youtube video describing different chemical mutagens and their function

Nice description about the mutagens (Youtube)

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