146 ◾ Fundamental Food Microbiology
Electrotransformation
In this method, a suspension of recipient cells in high population levels (10 8 cells/200 mL) is
mixed with purified DNA (1–2 μg) from a donor strain and then exposed to a high-voltage electric
field for a few microseconds. This results in temporary formation of small holes in the cell barrier
(membrane) through which purified DNA can pass. Subsequently, the cells are allowed to repair
their damage and express the new phenotype to enable their isolation.
This method has been widely used in many lactic acid bacteria to introduce plasmids from different strains of the same species as well as from separate species and genera. In addition, vectors
carrying cloned genes from diverse sources have been successfully introduced in several species of
lactic acid bacteria. This is currently the most preferred method to transfer DNA from a source
into recipient cells of lactic acid bacteria.
Conjugative Transposons
Transposons or transposable elements are segments of DNA in chromosomes or plasmids of bacteria
that can move from one site to another. Because of this, they can cause rearrangement in the sequence
of chromosomal and plasmid DNA and loss or gain of phenotypes in the hosts. The simplest transposons are known as insertion sequences (IS). Each IS element is an autonomous unit, containing
a coding region that has the gene encoding the transposase enzyme necessary for its transposition
on the host DNA and an inverted nucleotide sequence repeated at each end. Transposition of an IS
element occurs at target sites in host DNA, and after transposition, the host DNA contains short,
direct repeats on either end of an IS element. Larger transposons (Tn) contain different genes, such
as those that encode for an antibiotic-resistance phenotype in the central region and an IS element on
each end. Transposons can be conjugative and can be transferred from a donor to a host by the same
method used for a conjugative plasmid. When such a transfer occurs, the phenotypes encoded by the
resident genes (or genes cloned into it) are also transferred and expressed in a recipient strain.
A few IS elements and Tn have been identified in some strains of lactic acid bacteria. Lactobacillus
casei S1 contains the insertion element IS1, which has two ORFs and an inverted repeat at either
end. It is chromosomally located, and, when inserted in a prophage, the lysogenic phage becomes a
virulent phage. The nisin-producing Lac. lactis ssp. lactis ATCC 11454 contains a conjugative transposon, Tn5276, in the chromosome, which is approximately 70 kb and contains in the central region
the genes necessary for nisin A production (nis operon) and sucrose utilization. In addition, some
conjugative transposons from several Streptococcus and Enterococcus species that encode antibioticresistance markers, such as Tn916 (which encodes tetracycline resistance), have been transferred to
different strains of lactic acid bacteria. They have also been used in genetic recombination studies
in lactic acid bacteria. Besides, transposons (Tn916, Tn1545, Tn5) are also routinely used for gene
disruption to study the role of various virulence factors in pathogenesis for foodborne pathogens.
Gene Cloning
To transfer and express a gene from a donor into a recipient, the usual procedure is to clone
the gene into a suitable plasmid or cloning vector and introduce the vector into the recipient
cells. In the simplest form of this technique, a DNA segment carrying the genes is obtained by
digesting the purified DNA of the donor with the suitable DNA restriction endonucleases and
purifying it from the mixture. A suitable plasmid (cloning vector) that has one or several gene
Electrotransformation
In this method, a suspension of recipient cells in high population levels (10 8 cells/200 mL) is
mixed with purified DNA (1–2 μg) from a donor strain and then exposed to a high-voltage electric
field for a few microseconds. This results in temporary formation of small holes in the cell barrier
(membrane) through which purified DNA can pass. Subsequently, the cells are allowed to repair
their damage and express the new phenotype to enable their isolation.
This method has been widely used in many lactic acid bacteria to introduce plasmids from different strains of the same species as well as from separate species and genera. In addition, vectors
carrying cloned genes from diverse sources have been successfully introduced in several species of
lactic acid bacteria. This is currently the most preferred method to transfer DNA from a source
into recipient cells of lactic acid bacteria.
Conjugative Transposons
Transposons or transposable elements are segments of DNA in chromosomes or plasmids of bacteria
that can move from one site to another. Because of this, they can cause rearrangement in the sequence
of chromosomal and plasmid DNA and loss or gain of phenotypes in the hosts. The simplest transposons are known as insertion sequences (IS). Each IS element is an autonomous unit, containing
a coding region that has the gene encoding the transposase enzyme necessary for its transposition
on the host DNA and an inverted nucleotide sequence repeated at each end. Transposition of an IS
element occurs at target sites in host DNA, and after transposition, the host DNA contains short,
direct repeats on either end of an IS element. Larger transposons (Tn) contain different genes, such
as those that encode for an antibiotic-resistance phenotype in the central region and an IS element on
each end. Transposons can be conjugative and can be transferred from a donor to a host by the same
method used for a conjugative plasmid. When such a transfer occurs, the phenotypes encoded by the
resident genes (or genes cloned into it) are also transferred and expressed in a recipient strain.
A few IS elements and Tn have been identified in some strains of lactic acid bacteria. Lactobacillus
casei S1 contains the insertion element IS1, which has two ORFs and an inverted repeat at either
end. It is chromosomally located, and, when inserted in a prophage, the lysogenic phage becomes a
virulent phage. The nisin-producing Lac. lactis ssp. lactis ATCC 11454 contains a conjugative transposon, Tn5276, in the chromosome, which is approximately 70 kb and contains in the central region
the genes necessary for nisin A production (nis operon) and sucrose utilization. In addition, some
conjugative transposons from several Streptococcus and Enterococcus species that encode antibioticresistance markers, such as Tn916 (which encodes tetracycline resistance), have been transferred to
different strains of lactic acid bacteria. They have also been used in genetic recombination studies
in lactic acid bacteria. Besides, transposons (Tn916, Tn1545, Tn5) are also routinely used for gene
disruption to study the role of various virulence factors in pathogenesis for foodborne pathogens.
Gene Cloning
To transfer and express a gene from a donor into a recipient, the usual procedure is to clone
the gene into a suitable plasmid or cloning vector and introduce the vector into the recipient
cells. In the simplest form of this technique, a DNA segment carrying the genes is obtained by
digesting the purified DNA of the donor with the suitable DNA restriction endonucleases and
purifying it from the mixture. A suitable plasmid (cloning vector) that has one or several gene