Fundamental Food Microbiology, Third Edition - Fuad Fathir

160 FUNDAMENTAL FOOD MICROBIOLOGY
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.
F. 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) is also transferred
and expressed in a recipient strain. 3
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 nisinproducing
Lac. lactis ssp. lactis ATCC 11454 contains a conjugative transposon, Tn
5276, in the chromosome, which is ca. 70 kb and contains in the central region the
genes necessary for nisin A production (nis operon) and sucrose utilization. 7 In
addition, some conjugative transposons from several Streptococcus and Enterococcus
species that encode antibiotic-resistance markers, such as Tn 916 (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.
IV. 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 in this technique, a DNA segment,
carrying the genes, is obtained by digesting the purified DNA of the donor with the