Techniques For Insertional Mutagenesis In Mycobacterium Studies Biology Essay

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Functional studies of the Mycobacteria have been hindered by the difficulty to introduce mutations in the genomes of the various species that are found in the genus Mycobacterium. This problem can be overcome by making use of insertional mutagenesis techniques. This method of mutagenesis generation can be divided into two groups of techniques, transposon mutagenesis and allelic exchange. With the use of these techniques random and specific site mutations can be obtained, respectively. The results obtained from the use of these methods can be used to generate large mutation libraries and even be used in the generation of new vaccines and novel medications in the fight against disease causing organisms from this genus.

The use of molecular genetic techniques has revealed much about the genus Mycobacterium [1],[2]. The study of these organisms is important in cases of the disease causing mycobacterial species such as Mycobacterium leprae (which causes leprosy), M.tuberculosis (which causes tuberculosis) and M. avium (which causes infection in immune compromised individuals) [1-3].

The availability of sequenced genomes has furthered our knowledge into organisms. Techniques for the study of genomes include the insertional mutagenesis techniques, which will be discussed in this review. Insertional mutagenesis is the use of a marker gene to select for a mutagenic event [4]. These techniques can be divided into two groups, transposon mutagenesis techniques and allelic exchange techniques. Illegitimate recombination will not be discussed as a technique because it is randomly occurring within the cell.

Transposon mutagenesis techniques make use of transposable elements that have the ability to relocate within the genome of an organism [5],[6]. These transposable elements can insert themselves into random positions in the genome or at insertion hot spots [7]. This technique allows for the creation of mutation libraries that can be used in the production of new vaccines and medicines [8].

Allelic exchange techniques are complex and allow for the generation of specific mutations within organisms with the use of the natural occurring process, homologous recombination. These specific mutations are important in assigning a phenotype to a gene of interest or to determine if a gene or a gene cluster is necessary for the survival of the organism. The use of these techniques has one major drawback and that is the high frequency of illegitimate recombination that occurs [6].

It has however been difficult to study species from the genus Mycobacterium with the use of molecular genetic techniques. Mycobacterial studies are hindered by the slow growth of these organisms and some physical properties, including the hydrophobic nature of the cell wall that causes the cells to clump and the difficulty in penetration of the cell wall [9],[10].

Insertional mutagenesis techniques are attractive for the creation of mutations in mycobacteria because they overcome the limitations of the DNA-damaging agents that may create more than one mutation in the genome of a single organism [7]. Other techniques such as conjugation, transduction and transformation are hindered by the cells growing in clumps or the slow doubling time of some species, such as M. tuberculosis which has a doubling time of sixteen hours under optimal conditions [5],[11].

Over the past two decades insertional mutagenesis techniques have been developed and improved upon. This has increased our knowledge of the mycobacterium genome and the pathogenesis of some species. The knowledge gained has had and will have great implications for the development of new and better vaccines as well as novel medications. These techniques will now be discussed.

2. Transposon Mutagenesis in Mycobacteria

Transposon mutagenesis is a method that makes use of a transposable element to create random or specific-site mutations with its insertion into genome [12]. With the disruption of a gene, the gene expression should be altered and the resulting phenotype can indicate the function of that gene. This technique can be used to create large mutation libraries and to investigate virulence factors [10].

A large amount of transposable elements have been identified in mycobacteria and can be used in transposon mutagenesis studies, some of these include: IS1096 originating from M. smegmatis and IS6110 from M. tuberculosis [5],[8].

2.1 The use of thermosensitive and antibiotic resistant vectors as a transposon delivery system

Guilhot et al. (1994) created the first mycobacterial insertional libraries. The transposon was inserted directly into the genome of the mycobacterium with the use of a constructed plasmid [8].

With the use of the pCG79 (streptomycin-spectinomycin and kanamycin resistant) plasmid M. smegmatis was transformed. This is a non-replicative plasmid with a mutation that renders it thermosensitive for replication. Cells transformed with the pCG79 plasmid were grown on plates that contained kanamycin. This plasmid contains a Tn611 transposable element that integrates itself into the genome of the mycobacterium. With the use of Southern hybridization the cells were checked to determine if the kanamycin and thermosensitive properties obtained were as a result of transposition [8].

Results obtained showed that most of the colonies obtained were as a result of the transposition of the Tn611 element into the genome of the organisms. This transposition occurred at random and the colonies showed different insertion sites upon hybridization. Some of the colonies obtained showed the same banding pattern. These colonies were the result of replication occurring before the cells were plated out and not because of potential insertion hot spots being present within the genome. Some of the colonies obtained were as a result of illegitimate recombination. With the use of the pCG79 plasmid it is possible to create large amounts of mutants that originated due to the transposition of the Tn611 element [8].

The low rate of electroporation efficiency and the low rate of transposition is a problem for production of large mutation libraries [10]. With the use of a replicating vector the problem of low transformation can be overcome when combined with a counterselectable marker. The counterselectable marker selects for the colonies that no longer have the vector present within the cell, by causing the death of these cells [12].

Guilhot et al.(1994) developed a system where the conditionally replicating vector was lost at 39°C [13]; however this cannot be applied to the slow growing mycobacterial species such as M. tuberculosis because in these organisms the thermosensitive vectors are not as effective as in the fast growing species [12].

Cirillo et al. (1991) described the first system that made use of the mycobacterial thermosensitive origin of replication as well as the counterselectable marker, sacB to select for mutants gained by insertional mutagenesis [14]. Pelicic et al. (1997) determined that the use of both the counter selectable marker and the thermosensitive origin of replication delivered a high efficiency of counter selectability. Thermosensitivity was weaker in M. tuberculosis than in M. smegmatis, but the counterselection was higher in M. bovis BCG than in M. smegmatis. It is for this reason that it is believed the dual selection is so very efficient. This system was used to create the first transposon mutagenesis libraries for M. tuberculosis [6].

Transpositions should occur in a conservative manner, this allows for the positive selection of colonies that had undergone insertional mutagenesis. The counterselectable marker sacB, which originates from Bacillus subtilis, is lethal when expressed in mycobacteria cultured on a media containing sucrose [15-17]. The ts-sacB vectors used by Pelicic et al. (1997) were successfully used in the transformation in M. tuberculosis and M. bovis BCG. It was also determined that the ts-sacB vectors could also be used in studies of allelic exchange [6].

2.2 The use of mycobacteriophages as a transposon delivery system

Jacobs et al. (1987) created the first recombinant shuttle phasmids, from an E. coli cosmid that was inserted into the DNA of the mycobacteriophage. These shuttle phasmids had the ability to replicate in E. coli as plasmids and in mycobacteria as phages. They were the first shuttle vectors that allowed the insertion of foreign DNA into the genome of the mycobacteria by infection [18].

In 1988 Snapper et al. published results on the generation and expression of selectable genetic markers in mycobacteria, in their studies they made use of a cosmid that originated from Escherichia coli and inserted it into the genome of the mycobacteriophage L1. This resulted in the formation of a shuttle phasmid which replicated as a plasmid in E. coli. The shuttle phasmid and the phage both had the ability to lysogenize the mycobacteria [19].

Bardarov et al. (1997) developed a transposon mutagenesis system that created conditionally replicating shuttle phasmids from mycobacteriophages, D29 and TM4. These mycobacteriophages are a very efficient delivery system for both the fast and slow growing mycobacterial species. They have the added advantage of being able to potentially infect every cell present in the population and create large numbers of mutations resulting in large mutation libraries [7].

The construction of the conditionally replicating shuttle phasmid from mycobacteriophages were done with the use of a cosmid that originated from E. coli and possessed either a Tn10 (kan) or Tn5367 transposon. This cosmid was inserted into the non-essential part of the mycobacteriophage genome. Thermosensitive mutations were created in the genomes of the mycobacteriophages so that replication could occur at 30°C for both phages but not at 37°C for the TM4 phage and at 38.5°C for the D29 phage [7].

It was determined that infections with the shuttle phasmids that contained the Tn5367 transposon had random insertions in the genome and cells that were infected with the shuttle phasmids that contained the Tn10 (kan) transposon, had insertions in specific sites [7].

The use of nonreplicating vectors has low transformation frequencies with electroporation in comparison to that of the conditionally replicating shuttle phasmids. One disadvantage of the conditionally replicating vectors is that without the regulation of the transposition event there cannot be selected against the propagation of transposon mutants in the outgrowth phase. The effect of this is that many of the selected mutants will be siblings. This problem can be overcome by selecting the mutants shortly after infection, because with the use of conditionally replicating phages, nearly every cell in the culture will be infected. The result being that mutations will be chosen to represent individual insertion events that has occurred within the organism [7].

2.3 STM - Signature Tagged Mutagenesis

Signature tagged mutagenesis (STM) has great implications in studies of pathogenic organisms because can be used to identify the genes in these organisms that make them virulent. Identification of these genes is achieved with the inoculation of mutant organisms, which are labelled with a specific genetic tag, into a susceptible host [20]. A large number of mutants are tested in one host animal at the same time after which identification is done on the mutants that failed to colonize in the host tissues. These mutants can be detected with the use of PCR and DNA hybridization techniques [21].

This technique is useful in determining the genes that are essential for a pathogen to cologenize in the host organism and has been used to screen the mutant library of M. tuberculosis [20].

In a study done by Collins et al. (2005), they combined illegitimate recombination with STM in comparison to the previous studies where they used the mutation libraries that were generated with the use of transposon mutagenesis studies. They determined that illegitimate recombination can be used as a screening method because of the identification of a block of genes that might contribute to the virulence of the organism [21].

In transposon mutagenesis studies the insertion of the transposable element into the DNA of the organism without any deletions occurring. With illegitimate recombination a deletion of DNA at the site of insertion occurs that may cause the deletion of multiple genes. The combination of illegitimate recombination with STM holds great promise for studies in developing new vaccines against pathogens because of these deletions that occur [21].

STM is limited by the small size of each mutant pool that can be used in these studies as well as being very labour intensive in the performing of the comprehensive screens, however this is a very valuable technique [22].

2.4 TraSH - Transposon Site Hybridization

Transposon site hybridization (TraSH) is a combination of transposon mutagenesis and microarray hybridization. It can be used to determine the genes that are presumed to be essential for the organisms of study under certain environmental conditions [22]. The knowledge gained from these experiments is useful in the development of novel drugs, especially the studies of M. tuberculosis.

The technique makes use of a DNA microarray that contains a fragment of DNA derived from every presumed open reading frame in the genome of the organism. This is used to map all the transposon insertions that occur in a pool of mutants. The identification of locations of the transposon sites in the genome was obtained with the use of labelled RNA that hybridized to the chromosomal sequences adjacent to the transposon after the transposition occurred. The isolation of DNA from the mutants is followed by the digestion of the DNA with the use of restriction enzymes after which adapters are ligated to the fragments obtained. PCR using adapter- and transposon-specific primers were used to amplify the regions adjacent to the transposon insertion sites. This is then followed by transcription with the use of a RNA polymerase and the resulting RNA is then labelled and then hybridized to the DNA microarray [22].

Using this method, TraSH can be used to identify the region adjacent to the insertion of the transposon in the genome because the DNA microarray contains probes that bind to fragments of RNA that were obtained from the PCR reaction [22].

Sassetti et al. (2001) made use of auxotrophic mutants that are unable to cause disease and identified the mutated genes that are of known function as well as unknown function in hopes of generating an attenuated strain of M. tuberculosis [22].

The mycobacterial infection of M. tuberculosis presents in two stages, the acute infectious stage and the latent stage of the disease. Beste et al. (2009) made use of TraSH to determine the genes that are expressed during the slow growth of the bacteria and genes that are expressed when the organism switches from the slow growth to fast growth [11].


Allelic exchange is a method that can be used to generate specific chromosomal mutations and this is achieved by homologous recombination and the use of a vector that contains a marker gene(s) being flanked by a piece of upstream and downstream DNA. Through homologous recombination this vector is inserted into the genome of the organism of interest with a single cross over event (SCO) [23].

To achieve homologous recombination in slow growing mycobacterial species is difficult because of the amount of illegitimate recombination that occurs as well as the nature of the recA gene in these organisms [24].

Since Kalpana et al. (1991) demonstrated illegitimate recombination in slow-growing species of mycobateria at a relatively high rate and it has also been shown that illegitimate recombination occurs at a low rate in the fast-growing species [25],[26]. This occurrence is a problem in allelic exchange studies when the rate of illegitimate recombination exceeds that of homologous recombination [23]. The pre-treatment of DNA with either UV light or alkali has had some success in stimulating homologous recombination, however boiling has not [25],[23].

The recA protein is a functional protein in homologous recombination, M. tuberculosis and M. leprae recA protein contains intein which is removed by protein splicing. M. smegmatis does not have intein in its recA protein and homologous recombination occurs more frequently within this organism. The mechanism by which recA protein functions in homologous recombination is not yet known. [24].

3.1 Introduction of foreign DNA into the genome of mycobacteria with the use of homologous recombination

Husson et al. (1990) achieved allelic exchange through homologous recombination by using a shuttle vector that replicates in E. coli but not in M. smegmatis. The shuttle vector used, pY6001, contains an ampicillin resistance gene (Apr) and the M. smegmatis pyrF gene. The gene allows for the integration of the vector into the genome through homologous recombination, but it also acts as both a positive and negative control [27].

Positive and Negative selection is possible because the pyrF gene encodes orotidine monophosphate decarboxylase. The gene allows the growth of the organism on a media that does not contain uracil and no growth of cells on a media that contains 5-FOA [28].

With the introduction of the amp gene into the pY6001 vector, the pY6002 vector was created that conveys kanamycin resistance and also disrupts the pyrF gene. The colonies obtained from transformation with pY6002 could be divided into two classes based on the physical properties of the transformed cells. Class I colonies were the presumed products of a single homologous recombination event due to the presence of both a functional and a disrupted pyrF gene in the genome. They also possessed physical properties such as being 5-FOA sensitive and being uracil prototrophic organisms. The organisms that were found to be class II organisms had only the disrupted pyrF gene present in the genome and were also found to be resistant to 5-FOA. These organisms were also found to be uracil auxotrophic organisms as they could not synthesize their known requirements from the substrate uracil. Class II mutants were considered to be the result of a second homologous recombination event that had occurred and not the deletion of the duplicated genes that were present in the genome after the SCO [27].

Allelic exchange in M. tuberculosis with the use of short linear DNA fragments has not been successful, but has been achieved by making use of long linear recombination substrates, with about 20kb flanking the mutated gene on each side [4]. The success of recombination with the pyrF gene could be attributed to the long linear fragments of DNA flanking the gene while the other contributing factors for homologous recombination events remains unknown.

It is for this reason that methods for the pre-treatment of DNA was created to increase the frequency of homologous recombination occurring relative to the amount of illegitimate recombination that occurs within the organism [25].

Balasubramanian et al. (1996) created an excisable cosmid that allows the release of a large piece of mycobacterial DNA from the recombinant cosmid. The introduction of the mutated allele into the cosmid was achieved with the use of homologous interplasmid recombination in E. coli. This is done by the subcloning of a fragment that contains the gene of interest followed by the insertion of a marker gene into the cloned gene. The mutated gene of interest that contains the marker gene is then inserted into the cosmid by homologous recombination. Transformation of the M. tuberculosis cells was achieved by electroporation and then the cells were plated onto media containing amino acids for growth of leucine auxotrophs generated by allelic exchange as well as any other auxotrophs generated by illegitimate recombination. They determined that all the auxotrophs generated were as a result of homologous allelic exchange as all of them were found to require leucine for growth [4].

In studies making use of long circular recombination substrates an equal amount of transformants were obtained however, only studies making use of long linear recombination substrates had auxotrophic mutants as a result [4].

Gene replacement with the use of short linear substrates within E. coli could not occur because of the overactive exonuclease activity, the effectiveness of their recombination enzymes and the need for recombination to be induced. The use of long linear substrates in these organisms has the advantage of being degraded slowly as well as possessing the essential recombination junctions [4].

Pashley et al. achieved gene replacement in mycobacteria with the use of two replicating plasmids that are incompatible and compete with each other during replication and division into daughter cells. These plasmids have similar replication proteins and as such are able to grow together in the presence of an antibiotic, but the removal of the antibiotic causes the death of one or both of these vectors. This creates the possibility for different kinds of selections that could have different copy numbers of the plasmids as a result. The use of selection can generate large numbers of the plasmid that contains the mutated allele or gene fragment. These vectors are easier to lose in slow growing mycobacteria because of selection than the thermosensitive vectors in these organisms [29].

Studies that make use of non-replicating vectors, long linear DNA fragments and incompatible plasmids usually make use of a counter selectable marker like sacB and a transducing shuttle plasmid. These studies have an amount of transformation and selection that needs to occur making these experiments lengthy, they also require large amounts of DNA (1-10 µg) and tend to yield low amounts of mutant colonies [30].

3.2 Recombineering in mycobacteria

Recombineering is a technique that can be defined as genetic engineering, making use of recombination proteins to promote homologous recombination in the host cell [31], [30]. Recombineering was developed in E.coli with the use of phage recombination systems [32].

The Rac prophage encodes the RecE and RecT proteins which encodes a double stranded DNA (dsDNA) dependent exonuclease and a single stranded DNA (ssDNA) binding protein respectively and are functional analogs of the Exo and Beta proteins found in the λ red system [33],[32]. These proteins are useful in studies that make use of recombineering as they only require short lengths of homologous DNA (50 bp) to promote homologous recombination at a higher level than is usually found within the cell [30].

As the mycobacterial species have a low amount of recombination occurring within these organisms, recombineering has been useful in the promotion of homologous recombination. Kessel et al. (2007) identified a mycobacteriophage that had similar proteins to that found in the Rac prophage. Mycobacteriophage Che9c was shown to achieve allelic exchange in both the fast- and slow-growing mycobacterial species. The mycobacteriophage encodes the proteins gp60 and gp61, which act as a dsDNA exonuclease and a ssDNA binding protein, respectively. Kessel and Hatfull (2007) also determined that both of these proteins had to be present to promote homologous recombination in the host organism [30].

Kessel et al. (2008) made use of mycobacterial strains that had an extra-chromosomal plasmid present within the cell. These plasmids, pJV53 and pJV62 are used for dsDNA and ssDNA recombineering, respectively. These plasmids have an acetamidase promoter present that controls the expression of the phage recombination genes, gp60 and gp61 from mycobacteriophage Che9c. The plasmid pJV53 contains genes 60 and 61 for recombineering of dsDNA and plasmid pJV62 only contains the gene 61 for ssDNA [31].

The construction of the targeted gene replacement in these recombination studies is achieved by die electroporation of linear dsDNA substrates into the mycobacterial strains that contain the extra-chromosomal plasmids for the promotion of homologous recombination [31].

The construction of point mutations with recombineering makes use of ssDNA substrates. This is because the frequency of recombination is higher with the use of ssDNA than when compared to dsDNA. The choice of which of the genomic strands to target during recombineering is also very important as it could have an effect on the efficiency of recombination. Substrates with a length of 48 nucleotides show optimal recombination frequencies. A method to make sure that recombination has occurred is to introduce an oligionucleotide substrate that introduces the chromosomal point mutation that brings about drug resistance [31].

Recombineering could as such be viewed as a potentially invaluable technique in the future of mycobacterium genomic research and because this technique's use in mycobacteria is still new, it could still be much improved.

4. Conclusion

Studies making use of insertional mutagenesis techniques, such as the techniques mentioned in this review, has been invaluable in the studies of mycobacteria, especially the pathogenic mycobacteria that have a large impact on the lives of many people that are infected with these organisms.

The use of transposon mutagenesis and the creation of mutation libraries have had many applications and are still used today. These methods that employ the transposable elements found within the genome of the mycobacteria were used to create random mutations within the genome of the study organisms to potentially identify the functions of genes. Transposon mutagenesis study techniques are still being improved and created as can be seen with techniques such as STM and TraSH.

Allelic exchange studies have greatly been hindered by die frequencies of illegitimate recombination that occurs in the mycobacterial species, especially the slow growing species such as M. tuberculosis. With the use of phagemid DNA in recombination studies with M. smegmatis, no illegitimate recombination has been observed and similar results were obtained in studies with M. intracellulare and M. tuberculosis [25]. This overcomes the problem of the great frequency at which illegitimate recombination occurs and achieves the production of allelic exchange mutations. These include gene knockouts, point mutations, deletions and small insertions [31]. Allelic exchange methods are still being created and improved as can be seen with the recently developed recombineering technique.

Table 1

Comparison of transposon mutagenesis techniques and allelic exchange techniques.









Shuttle plasmids







Thermosensitive vectors



Antibiotic resistant vectors



Conditionally replicating vector


Counterselectable markers





Homologous recombination


Phage recombination systems



Randon mutations/Hot spots


Specific mutations



Many of the improvements in techniques that make use of transposon mutagenesis have been applied to techniques that make use of allelic exchange methods. It can thus been said that these methods have evolved together over the last couple of decades.

The creation of mutation libraries, knockouts, auxotrophic mutants, point mutations etc. in virulence studies have greatly impacted the understanding of how these organisms function in their pathogenesis and survive within host organisms. It has been applied to the potential creation of new and better vaccines and medications. It is for this reason that the continued development of these techniques is important.