Genetics of Some Beneficial Traits ◾ 145
a phenotype, the transconjugant will have the phenotype. To make DNA transfer possible, the
donor cells should have several other genes, such as a clumping factor (for physical contact through
clumping) and a mobilizing factor (to enable the DNA to move from a donor to a recipient). The
process consists of selecting the right donor and recipient strains, mixing the two cell types in a
donor:recipient ratio of 2:1 to 10:1 in several different ways for DNA transfer to occur, and then
identifying the transconjugants by appropriate selection techniques.
This technique has been used successfully to transfer several plasmid-linked phenotypes in
some lactic acid bacteria. The plasmid-linked Lac + phenotype was transferred conjugally between
two Lac. lactis species. The transconjugant was Lac + and had the specific plasmid, the loss of which
resulted in its phenotype becoming Lac – . Subsequently, the Lac + phenotypes located in different
plasmids in many Lac. lactis subspecies and strains were conjugally transferred to Lac – strains of the
same species. Conjugal transfer of different plasmid-linked traits has also been reported in other
lactic acid bacteria, such as the diacetyl production trait in Lac. lactis ssp. lactis biovar diacetilactis.
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., pAMβ1, 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.
Transformation
This 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 have 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.
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.
a phenotype, the transconjugant will have the phenotype. To make DNA transfer possible, the
donor cells should have several other genes, such as a clumping factor (for physical contact through
clumping) and a mobilizing factor (to enable the DNA to move from a donor to a recipient). The
process consists of selecting the right donor and recipient strains, mixing the two cell types in a
donor:recipient ratio of 2:1 to 10:1 in several different ways for DNA transfer to occur, and then
identifying the transconjugants by appropriate selection techniques.
This technique has been used successfully to transfer several plasmid-linked phenotypes in
some lactic acid bacteria. The plasmid-linked Lac + phenotype was transferred conjugally between
two Lac. lactis species. The transconjugant was Lac + and had the specific plasmid, the loss of which
resulted in its phenotype becoming Lac – . Subsequently, the Lac + phenotypes located in different
plasmids in many Lac. lactis subspecies and strains were conjugally transferred to Lac – strains of the
same species. Conjugal transfer of different plasmid-linked traits has also been reported in other
lactic acid bacteria, such as the diacetyl production trait in Lac. lactis ssp. lactis biovar diacetilactis.
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., pAMβ1, 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.
Transformation
This 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 have 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.
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.