Fundamental Food Microbiology, Third Edition - Fuad Fathir

GENETICS OF SOME BENEFICIAL TRAITS 159
The method has several limitations, some of which have been listed previously
with the characteristics of plasmids. They include plasmid size, plasmid incompatibility
and instability in recipient strains, inability to express in hosts, inability to
have proper donors and recipients, and, in some cases, inability to recognize the
transconjugant. However, by using a broad-host-range plasmid (e.g., pAMb1, a
plasmid of Enterococcus spp. encoding an antibiotic gene), it was shown that plasmid
transfer by conjugation is possible among lactic acid bacteria between the same
species, between two different species in the same genus, or even between two
different species from different genera.
C. Transformation
The method involves extraction and purification of DNA from a donor bacterial
strain and mixing the purified DNA with the recipient cells. Some DNA fragments
encoding a phenotype are expected to pass through the cell barriers (wall and
membrane) and become part of the host DNA expressing the new phenotype. In
some Gram-positive bacteria (e.g., Bacillus spp.), this method has been effective to
transfer certain traits. In lactic acid bacteria, limited studies revealed that the technique
was not very effective; however, a modified method was effective. First, Lac.
lactis cells were treated with lysozyme or mutanolysin, or both, to remove the cell
wall and to form protoplasts in a high osmotic medium. The protoplasts were then
exposed to purified DNA (chromosomal, plasmid, or phage DNA) in the presence
of polyethylene glycol. The growth conditions were then changed for the protoplasts
to regenerate the cell wall. The transformants were then detected in a selective
medium. By this method, Lac + phenotype and Em r (erythromycin-resistance phenotype)
and phage DNA (transfection) were transferred to recipient strains of Lac.
lactis subspecies. Because of limitations in the success rate, this method is not widely
studied in lactic acid bacteria.
D. Protoplast Fusion
The technique involves preparation of protoplasts of cells from two different strains
and allowing them to fuse together in a suitable high-osmotic environment. Fusion
of cells of the two strains and recombination of the genetic materials may occur. By
allowing the protoplasts to regenerate the cell wall and by using proper selection
techniques, recombinants carrying genetic information from both strains can be
obtained. The technique has been used successfully to produce recombinants of Lac.
lactis subspecies for both Lac + and Em + phenotypes. However, because of the low
success rate, this technique is not used much in lactic acid bacteria.
E. Electrotransformation
In this method, a suspension of recipient cells in high population levels (10 8 cells/200
ml) is mixed with purified DNA (1 to 2 mg) 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
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