5]. Recombination techniques have been shown to occur at a very
low frequency in mycobacteria and because of this a plasmid-based
two-step strategy has been found to be more successful in obtaining
mutant strains [4], see Note 1. The first step involves the construction of a single crossover strain (SCO); this is then grown without
antibiotics to allow the second crossover event to occur, resulting in
a double crossover strain (DCO). The double crossover event can
lead to two outcomes, either the gene of interest is deleted or the
wild-type background is restored. Suicide vectors lack a mycobacterial origin of replication and are pretreated with either
UV-irradiation or alkali prior to transformation, to stimulate
homologous recombination [6]. To create an unmarked mutant,
a suicide vector based on a p1NIL or p2NIL backbone, with the
hygR, lacZ, sacB cassette from pGOAL19, is constructed to carry
approximately 1 kb of flanking DNA from the gene of interest
[4]. The final suicide vector construct contains selectable antibiotic
(hygromycin and kanamycin) markers; both are required since the
frequency of spontaneous mutation in mycobacteria can be equal to
the frequency of recombination. The lacZ gene is included to act as
a visible indicator of transformants which carry the plasmid, which
allows SCO and DCO transformants to be identified; and sacB,
which confers sucrose sensitivity, DCOs which have lost the suicide
vector will therefore be able to grow on media-containing sucrose
and will appear white in color [4].
However, DCOs are only obtained when the gene of interest is
nonessential. Since the deletion of essential genes generates a lethal
phenotype, it stands to reason that it is difficult to identify an
essential gene. The classical method of determining essentiality is
to introduce a second copy of the gene of interest and show that the
chromosomal copy can be deleted in this merodiploid strain, e.g.,
when a functional copy of the gene resides elsewhere. Replicating
plasmids, which express genes under the control of either a constitutive promoter, such as hsp60; or an inducible promoter, such as
the acetamidase promoter region from Mycobacterium smegmatis
[7], and the tetracycline inducible P TET system [8], can be used to
express the functional gene copy. However, problems with stability
have been reported, especially when introduced into Mycobacterium tuberculosis, which make these systems unreliable [9–12]. To
overcome this, a different type of vector has been created, integrating vectors, which merge into the chromosome. Mycobacterial
integrating vectors typically contain the mycobacteriophage L5
integrase which catalyzes site-specific recombination between attP
held on the plasmid and the chromosomal attB site [13]. With the
second gene copy in place, double crossovers (DCOs) can be
obtained by homologous recombination which have only the functional, integrated copy present, these are termed delinquents (Δ
0 int
alleles), i.e., carrying both deletion and integrated functional
alleles. To confirm the requirement of this integrated copy, the
286
Amanda Claire Brown
low frequency in mycobacteria and because of this a plasmid-based
two-step strategy has been found to be more successful in obtaining
mutant strains [4], see Note 1. The first step involves the construction of a single crossover strain (SCO); this is then grown without
antibiotics to allow the second crossover event to occur, resulting in
a double crossover strain (DCO). The double crossover event can
lead to two outcomes, either the gene of interest is deleted or the
wild-type background is restored. Suicide vectors lack a mycobacterial origin of replication and are pretreated with either
UV-irradiation or alkali prior to transformation, to stimulate
homologous recombination [6]. To create an unmarked mutant,
a suicide vector based on a p1NIL or p2NIL backbone, with the
hygR, lacZ, sacB cassette from pGOAL19, is constructed to carry
approximately 1 kb of flanking DNA from the gene of interest
[4]. The final suicide vector construct contains selectable antibiotic
(hygromycin and kanamycin) markers; both are required since the
frequency of spontaneous mutation in mycobacteria can be equal to
the frequency of recombination. The lacZ gene is included to act as
a visible indicator of transformants which carry the plasmid, which
allows SCO and DCO transformants to be identified; and sacB,
which confers sucrose sensitivity, DCOs which have lost the suicide
vector will therefore be able to grow on media-containing sucrose
and will appear white in color [4].
However, DCOs are only obtained when the gene of interest is
nonessential. Since the deletion of essential genes generates a lethal
phenotype, it stands to reason that it is difficult to identify an
essential gene. The classical method of determining essentiality is
to introduce a second copy of the gene of interest and show that the
chromosomal copy can be deleted in this merodiploid strain, e.g.,
when a functional copy of the gene resides elsewhere. Replicating
plasmids, which express genes under the control of either a constitutive promoter, such as hsp60; or an inducible promoter, such as
the acetamidase promoter region from Mycobacterium smegmatis
[7], and the tetracycline inducible P TET system [8], can be used to
express the functional gene copy. However, problems with stability
have been reported, especially when introduced into Mycobacterium tuberculosis, which make these systems unreliable [9–12]. To
overcome this, a different type of vector has been created, integrating vectors, which merge into the chromosome. Mycobacterial
integrating vectors typically contain the mycobacteriophage L5
integrase which catalyzes site-specific recombination between attP
held on the plasmid and the chromosomal attB site [13]. With the
second gene copy in place, double crossovers (DCOs) can be
obtained by homologous recombination which have only the functional, integrated copy present, these are termed delinquents (Δ
0 int
alleles), i.e., carrying both deletion and integrated functional
alleles. To confirm the requirement of this integrated copy, the
286
Amanda Claire Brown