Auxotrophic mutants are bacteria, yeast, protoplast or mammalian host cell strains that can’t produce a nutrient vital for growth due to genetic mutations. As a result, these strains are unable to survive in media lacking that specific nutrient unless it is provided externally.
The term auxotrophy refers to a nutritional dependency, where the mutant requires an auxiliary or supplemental source of the missing nutrient for its growth and survival. In contrast, the corresponding wild type strain can synthesize that specific nutrient and is not dependent on an exogeneous supply in the growth medium.
Auxotrophic mutants are often used in genetic studies, selection experiments, and research involving metabolic pathways and nutrient utilization. They are also very useful in gene cloning procedures.
In this article, we will learn what auxotrophic mutants are, and how scientists utilize these mutant strains for research studies, especially in the context of gene cloning. We will also look at some commonly used bacterial and yeast auxotrophic strains widely used in research labs.
Article table of contents:
Mechanisms or genetic basis of Auxotrophy
Auxotrophic mutants in gene cloning
Why do researchers choose to use auxotrophic mutations over antibiotic selection?
Auxotrophic Agrobacterium tumefaciens strains
DL 39: An auxotrophicE. coli strain
What are auxotrophic mutants?
Auxotrophy is a phenotype of an organism where it cannot synthesize a particular compound/ metabolite required for its growth and is an auxotroph/ auxotrophic mutant for that compound.
An auxotroph is dependent on the medium for getting the supply of that corresponding necessary compound. If that culture medium lacks that compound, the auxotroph cannot survive.
Creating auxotrophic mutants is a common technique in microbiology and genetic engineering for various purposes, such as selecting transformed cells, and studying metabolic pathways.
The specific nutrients chosen will depend on the intended use of the mutant strain.
Some common examples of compounds that auxotrophic mutants cannot synthesize include amino acids (arginine, histidine, leucine, isoleucine, lysine, tryptophan, methionine), vitamins, nucleotides, cofactors and coenzymes (NAD, FAD).
Mechanisms or genetic basis of auxotrophy
An auxotrophic mutant’s inability to synthesize a particular nutrient is the result of mutations in the genes responsible for the biosynthesis of the compound, leading to the absence or dysfunction of the enzymes involved. The genetic basis of auxotrophy is rooted in the disruption of these biosynthesis pathways or enzyme functions.
Biosynthetic pathway deletion mutations:
Many essential compounds, such as amino acids, nucleotides, and vitamins are synthesized through complex biochemical pathways.
Deletion mutations in the genes encoding enzymes involved in these pathways can lead to the disruption of compound synthesis. As a result, the mutant organism cannot produce the compound, rendering it auxotrophic for that specific nutrient.
For example, consider an auxotrophic mutant that cannot synthesize the amino acid tryptophan (figure 1).
This could be due to a mutation in one of the genes responsible for the various steps in the tryptophan biosynthetic pathway. Without functional enzymes, the mutant cannot complete the synthesis of tryptophan and requires an external source of the amino acid for growth.
Figure 1. Demonstrates the growth results of an auxotrophic mutation for the amino acid tryptophan.
In figure 1, the auxotrophy lies in tryptophan synthesis. In the left panel, the normal, or wild type strain is shown. It grows on both rich media that has nutrients including tryptophan, and also on a media that lacks tryptophan because this strain can synthesize tryptophan.
In the right panel, the tryptophan auxotrophic mutant of the wild type strain is shown. It successfully grows on rich media that has an exogenous supply of tryptophan. However, when plated on media that does not have tryptophan, it fails to grow because it cannot synthesize tryptophan due to the auxotrophic mutation. If tryptophan is added back to this medium, this strain can grow.
To put things in perspective, the E. coli strain DL39, often used in gene cloning and expression studies, is an auxotrophic mutant for multiple amino acids including tryptophan.
Loss of enzyme function:
Auxotrophy may also arise from point mutations in a gene encoding an enzyme that is critical for the cell to synthesize a compound such as an amino acid. Here, the critical enzyme is present but is non-functional.
In this case, the enzyme is present in the host cell because the entire gene is not deleted, like we discussed in the previous section. However, it is nonfunctional because of point mutations that make it nonfunctional. These mutations alter one or more amino acids in the enzyme, that are critical.
To put things into perspective, enzymes are biological catalysts, facilitating specific chemical reactions. And mutations can cause structural and functional changes in enzymes, rendering them nonfunctional or significantly less active. Consequently, the affected biochemical reactions cannot proceed efficiently.
This is exactly the situation that we are discussing in this section. Consider an auxotrophic mutant that lacks functional enzymes required for converting a precursor molecule into a particular vitamin. In this case, the required enzymes are present, but a critical amino acid important for that enzyme’s catalytic capability has been changed to another amino acid by a point mutation in the enzyme’s gene.
Without the active enzymes, the conversion cannot occur and the mutant organism becomes dependent on an external supply of that vitamin.
Auxotrophic mutants in gene cloning
Auxotrophic mutations are extensively used in gene cloning and recombinant DNA technology. They provide a great strategy to select and identify cells that have successfully taken up foreign genes introduced by some horizontal gene transfer method such as transformation.
Here’s the workflow of how auxotrophic mutations are employed in gene cloning:
1. Selectable marker: Auxotrophic mutations act as selectable markers in cloning experiments. A strain with an auxotrophic mutation is unable to grow or survive without a specific nutrient (for example nutrient ‘A’ as shown in figure 2) that it cannot synthesize on its own.
Figure 2. Demonstrates how auxotrophic mutations are used as selectable markers in gene cloning experiments.
2. Transformation with foreign DNA:
Researchers introduce a plasmid or DNA fragment containing the gene of interest into the auxotrophic host cells. Foreign DNA often includes a functional copy of the missing gene that restores the biosynthetic pathway.
3. Complementation:
The introduced DNA provides a functional copy of the missing gene, allowing the host cell to produce the essential nutrient it couldn’t synthesize before.
4. Selection:
Cells that have successfully taken up and integrated the foreign DNA can now grow and reproduce in a minimal growth medium lacking the nutrient they were auxotrophic for. This creates a selective advantage for transformed cells because they can survive in conditions where non-transformed cells cannot.
5. Identification:
The growth of transformed cells on selective media indicates successful gene transfer and integration. This growth can be visually observed or measured so that researchers can identify and isolate transformed cells.
The transformed cells can be further cultured and propagated for downstream applications. The introduced gene can be expressed, producing the desired protein or molecule.
To sum up, auxotrophic mutations, enable positive selection of cells that have taken up foreign DNA, simplifying the process of gene cloning in that way.
Why do researchers choose to use auxotrophic mutations over antibiotic selection?
Researchers often choose to use auxotrophic mutations for several reasons when working with microorganisms in the laboratory:
Selective growth conditions: Auxotrophic mutants require specific nutrients for growth that are not present in minimal media. By supplying these nutrients in the growth medium, researchers selectively allow the mutant strain to grow while preventing the growth of wild type or non-mutant strains.
This provides a stringent and specific way to select desired mutants.
Cleaner genetic background: Antibiotic resistance genes, also known as antibiotic selectable markers, used for selection can sometimes interfere with downstream experiments or applications.
Auxotrophic mutants eliminate the need for antibiotic-resistance genes
in the construct, resulting in a cleaner genetic background for
subsequent studies.
Precision and specificity: auxotrophic mutants offer a high level of precision and specificity in selection. The requirement for a specific nutrient can be fine-tuned to achieve optimal growth conditions for the mutant strain while maintaining stringency against wild type or non-mutant strains.
Reduced interference: antibiotics used for selection can sometimes interact with other cellular processes or affect protein expression, leading to unintended effects. Auxotrophic mutants do not involve the introduction of external compounds like antibiotics that could potentially interfere with cellular functions.\
Minimized genetic load: introducing antibiotic resistance genes can increase the genetic load on the host organism, potentially affecting its growth rate or metabolic pathways. Auxotrophic mutants only require a targeted mutation in a specific biosynthetic pathway, minimizing the genetic burden.
No need for external agents: antibiotic selection requires the addition of antibiotics to the growth medium, which can be expensive and sometimes unstable over long periods of culture. Screening by capitalizing on auxotrophic mutant strains, eliminates the need for antibiotics – overcoming this issue.
Biosafety considerations: some studies, particularly those involving genetically modified organisms (GMOs) or environmental release, need to address biosafety concerns associated with antibiotic resistance genes. Also, it is always recommended to avoid, if possible, introducing antibiotic resistance in the strains (in the form of antibiotic selectable markers) you’re working on in the lab. For one, strains that do not have antibiotic resistance are safer for you to handle. Second, in case auxotrophic mutant strains leak out of the lab accidentally, they wouldn’t be of that much of a concern.
The use of auxotrophic mutants can alleviate these concerns by avoiding the introduction of antibiotic resistance traits into the environment.
Natural mimicry: auxotrophic mutants simulate natural conditions where certain microorganisms rely on specific nutrients available in their environment. This approach can be particularly relevant for understanding microbial behavior in ecological contexts.
It’s important to note that the choice between auxotrophic mutations and antibiotic selection depends your specific research goals, the characteristic of the microorganism, and your intended downstream applications.
Now that we've understood what auxotrophic mutant strains are, let’s look at a particular E. coli strain as an example.
DL 39: An auxotrophic E. coli strain
E. coli is the model lab bacterium for many purposes including serving as cloning and expression host strains. The E. coli strain DL 39 is an auxotrophic mutant for amino acids aspartic acid, isoleucine, leucine, phenylananine, tyrosine, and valine.
It serves as an expression strain, especially where these amino acids need to be fluorescently labelled and incorporated into the recombinant protein.
Reference
Collens et al. 2004. Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture. Biotechnol prog. 20(3): 890-6. doi: 10.1021/bp034306w.
El Malki, F., & Jacobs, M. (2001). Molecular characterization and expression study of a histidine auxotrophic mutant (his1−) of Nicotiana plumbaginifolia. Plant Molecular Biology, 45, 191-199.
Meng et al. 2014. Effects of antibiotics on in vitro-cultured cotyledons. In vitro cellular and developmental biology-Plant. 50 (436-441)
Prias-Blanco et al. 2022. An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation. Transgenic Res. 31(6): 661-676. doi: 10.1007/s11248-022-00328-4
Shackelford and Chlan. 1996. Identification of antibiotics that are effective 2 in eliminating Agrobacterium tumefaciens. Plant molecular biology reporter. 14 (50-57).
