Selection of Different Types of Mutants

In this article we will discuss about selection of different types of mutants:- 1. Resistant Mutants 2. Regulation-Defective Mutants 3. Auxotrophic Mutants.

1. Resistant Mutants:

From a wild-type bacterial population which is susceptible to agents like bacteriophage, various drugs, ultra-violet light etc., mutants which are resistant to any of these agents can be selected directly by using selective media. After induction of mutation with a suitable mutagen, the treated population is allowed to grow under permissive conditions for some time to allow the expression of the mutant gene.

The culture is then dilution-plated in a suitable selective medium which would be expected to completely suppress the growth of the wild type bacteria and would allow only the mutants to grow and form colonies. The mutant colonies can be picked up and purified by dilution plating to obtain pure clones of mutants.

For example, for isolation of resistant mutants against a specific lytic bacteriophage, the selective medium would be one containing the particular phage, so that the wild-type which is susceptible would be eliminated by the lytic phage and only the phage-resistant mutants would form colonies.

Purification is necessary to eliminate any wild-type cells that might be present associated with the resistant cells. Similar procedure can be adopted for selection of drug-resistant mutants, in which case the selective medium would be a growth-supporting one containing the drug at a concentration which is normally inhibitory for the wild-type cells.

Similar procedure can also be used for isolation of several other types of mutants. For example, mutations resulting in reversion of auxotroph’s to prototroph can be detected. If it is desired to obtain a prototroph (his⁺) from a population of histidine auxotroph (his^–), a mutagen-treated population of the auxotroph may be plated on a medium without histidine. Such a medium will allow growth of the prototroph only.

Another type of mutants that can be selected by a similar procedure is those having a wider substrate range in comparison to that of the wild type. This means that a mutant is capable of utilizing a particular substrate which the wild-type is unable to use. The mutagen treated population of the wild- type may be initially grown in a liquid medium containing that substrate.

As this medium would allow only the mutants to grow, an increase in the number of mutants would be expected (enrichment). Plating on the same selective medium would allow the growth of mutants which are capable of utilizing the substrate, while the wild-type cells will fail to grow, because of their inability to utilize the substrate.

2. Regulation-Defective Mutants:

The metabolic pathways are controlled by regulatory mechanisms. One of the important mechanisms is feed-back control of biosynthetic pathways in which the end-product inhibits the first enzyme of the sequence. The first enzyme in such case is an allosteric protein which can bind both the substrate and the end-product at different sites of the molecule.

Mutation may result in the formation of a defective enzyme protein with loss of regulatory function. Such regulation-defective mutants may arise spontaneously, or may be induced by treatment with mutagens. A well-known example is a naturally occurring defective mutant of Corynebacterium glutamicum which produces large quantity of glutamic acid and has been used for commercial production of this amino acid.

Regulation-defective mutants of bacteria can be detected by using specific antimetabolites in selection medium. An antimetabolite is generally a non-biological chemical compound which is a structural analogue of a metabolite. They are able to inhibit growth by stopping the synthesis of the metabolite by reacting with the first enzyme of the biosynthetic pathway of the particular metabolite. This property of an antimetabolite can be made use of in detection of defective mutants which have the ability to escape the regulatory mechanism.

For detection of such regulation defective mutants, a mutagen-treated bacterial suspension containing 10⁸ to 10¹⁰ cells/ml is spread over a medium containing an inhibitory concentration of the antimetabolite (specific for the metabolite). After incubation, only a few colonies develop in the plates, because most of the bacteria fail to grow in presence of the antimetabolite. The colonies develop only from the small number of antimetabolite-resistant mutants.

However, all such resistant mutants may not be regulation-defective, because resistance may develop also by other means. The regulation-defective mutants can be identified in the plates by the presence of a halo of satellite colonies around the larger colonies of the mutants.

The satellite colonies develop because the mutant colony secretes the metabolite into the medium which feeds the wild-type bacteria to make them grow. The satellite colonies are smaller in size, because they start developing later when the mutants secrete enough metabolite to overcome the effect of the antimetabolite (Fig. 9.67).

3. Auxotrophic Mutants:

An auxotrophic mutation differs from others in producing a loss of ability to synthesise an essential metabolite. It may be one of the protein amino acids, a nucleic acid base, a vitamin etc. As a result, the mutant loses ability to grow in a minimal medium which sustains growth of the wild-type, and it obligately requires supplementation of the metabolite which it no longer can synthesise.

Obviously, a straight-forward detection of auxotroph’s is not possible in a selective medium. A laborious method would be to grow the mutagen-treated population in a medium which allows growth of both the wild-type and auxotroph’s (complete medium) and then test each and every colony for its ability to grow in a complete medium and in a minimal medium plate.

Considering an average mutation rate of 10^-6 to 10^-7, it can be realized that one million to 10 million colonies must be tested to find out a single auxotroph. However, treatment with mutagens can considerably increase the mutation rate. Even then, detection of auxotroph’s remains a laborious task.

Two techniques which are used in conjunction to minimize this labour are the enrichment of the mutants in the total population by treatment with penicillin and the replica plating:

(a) Penicillin Enrichment Technique:

This method makes use of the bactericidal property of penicillin which is restricted to the actively growing organisms. The antibiotic cannot destroy the non-growing or resting bacteria. Thus, under conditions which support growth of the wild-type bacteria only, penicillin kills them preferentially and the mutants which do not grow survive.

As a result, the proportion of mutants in the total population increases substantially making their detection and isolation easier. For this purpose, the mutagen-treated population is grown in a minimal medium containing a lethal dose of penicillin for a period during which the wild-type bacteria grow and are killed by the antibiotic.

The surviving bacteria having a higher proportion of mutants are centrifuged and washed to remove penicillin. They are suspended and plated on complete medium for detection of mutants by the replica plating technique. Penicillin treatment may have to be repeated to obtain the desired enrichment of the mutants.

(b) Replica-Plating Technique:

It is a simple but a very efficient method for detecting auxotrophic mutants. The technique developed by Lederberg makes it possible to test a reasonably large number of discrete colonies growing in a plate for the occurrence of mutants. Generally, 100 to 200 colonies growing in a single plate can be tested at a time, so that the labour and tediousness of examining each and every colony individually for detection of mutants are greatly minimized.

The basic outfit (Fig. 9.68) consists of a wooden or metallic cylindrical block with a diameter slightly less than that of a standard Petri dish, a metallic collar and several pieces of velveteen cloth. The bristles of the cloth act as individual inoculating needles. The components are sterilized before use. The velveteen is spread tightly over the block and kept in place by the collar.

The master plate containing complete medium with 100-200 discrete colonies among which the majority colonies are wild type and few mutants is inverted over the velveteen covered cylindrical block and lightly pressed to transfer the impression of the colonies on to the bristles of velveteen.

After removing the master plate, a blank minimal agar plate is similarly placed on the velveteen surface to transfer the impression of the master plate to it. On incubation, colonies of only the wild-type grow on the minimal agar surface.

When the two plates, i.e. the master plate and the minimal agar plate, are examined, it is observed that some colonies of the master plate are absent in the minimal agar plate indicating that they are probable mutants — because they grow in complete medium but fail to grow in the minimal agar.

These colonies are picked up from the master plate and purified by dilution plates Individual colonies are picked up from the dilution plates and tested for their growth requirement. Detection of the mutants by comparison of the master plate and the minimal agar plates can be facilitated by transfer of the colony images of the master plate on a photographic film. By correct positioning of the minimal agar plate against the negative and by illumination from bottom, it becomes easier to locate the mutant colonies which are absent in the plate.

A schematic representation of the master plate and a replica plate of minimal agar is shown in Fig. 9.69: