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الأحد، 24 فبراير 2019



Gene transfer between strains, species, and even genera of LAB can occur in vivo with the known natural mechanisms, physiological transformation, transduction, and conjugation. Although the significance of these mechanisms varies greatly among the different species, they contribute to the horizontal gene transfer in the natural habitats of LAB and they can be utilized in the genetic modification of LAB strains. However, for the actual recombinant DNA techniques, reliable in vitro gene transfer mechanisms are essential and have been adapted to the most important LAB.
A. Gene Transfer In Vivo

1. Physiological Transformation
Natural transformation, the first gene transfer mechanism described, was described by F. Griffith in LAB in 1929 in S. pneumoniae. Later DNA was shown to be the transforming principle  . Transformation by natural competence has been thoroughly studied in Bacillus subtilis , indicating that a number of competence genes need to be expressed for natural competence to take place.

 In dairy microorganisms, Møller-Madsen and Jensen first described a natural transformation system in a few strains of Lc. lactis by transferring between lactococci the ability to produce a malty flavor. This observation of natural competence has not been satisfactorily confirmed. Møller-Madsen later claimed that only a few strains of lactococci were able to transform by natural competence and that, unfortunately, these strains were lost (A. Møller-Madsen, personal communication). However, a few results indicate that the original observations were not artefacts. Knite  reported the transfer of mannitol and streptomycin resistance in lactococci by natural transformation.

 Sanders and Nicholson later showed that nonprotoplasted lactococci was able to take up plasmids as well as phage DNA if polyethylene glycol (PEG) was present. Finally, and most convincing, is the identification of the complete competence operons in the Lc. lactis IL1403 genome sequence. These observations indicate that lactococcal natural competance has to be reconsidered as a mode of gene transfer in lactococci.

2. Transduction

Transduction is the transfer of DNA between two strains by means of bacteriophage. Transduction was first demonstrated the early 1960s in Lactococcus by Elliker and coworkers,[159,160] where tryptophan independence and streptomycin resistance, respectively, was transferred by a virulent bacteriophage c2. In the 1970s McKay et al. showed transduction of chromosomal traits like maltose and mannose utilization as well as plasmidlinked traits like lactose utilization and proteinase activities using induced temperate phages for the transduction.High-frequency transduction of lactose metabolism was shown by McKay et al. in repeated transduction experiments. However, this was not a general phenomenon of plasmid transfer, since pAM b 1 was not transduced with high frequency in secondary transductions. Birkeland and Holo showed that carrying of the cohesive ends from the temperate bacteriophage f LC3 increased the transduction efficiency of plasmids approximately

                     Hoang-Dung TRAN and friends 1000-fold. Recently, Chandry et al. showed that the cos-site from the lytic phage sk1 also increases the transduction frequencies of plasmids. In addition to lactococci transduction has been demonstrated in Lb. gasseri ADH[ and in S. thermophilus.[168] Heller et al.demonstrated transduction in a fermented milk environment by S. thermophilus phages. Since the development in the early 1980s of transformation systems for LAB, there has been only limited interest in transduction. This is probably due to the expected limitations in host range of the temperate and virulent bacteriophages. However, host range (e.g., plaque formation) is not limiting for gene transfer by phage.

3. Conjugation
During conjugation, DNA is transferred from the donor cell to the recipient by direct cellto-cell contact. In gram-negative bacteria this contact can be mediated with structures called sex pili, but they apparently have no role in conjugation between gram-positive bacteria, such as LAB. Instead, sex pheromones, or substances produced by recipient cells promoting the synthesis of a cell aggregation factor by the donor, thus leading to the formation of donor-recipient pairs, are well known, especially among the enterococci. Pheromones, however, are not universal among gram-positive bacteria with conjugative genetic elements. Irrespective of the mechanisms of achieving cell-to-cell contact, the conjugative genetic elements must have certain highly conserved common structures, such as the origin of DNA transfer (oriT) and the various genes involved in the actual transfer event (tra).

 The importance of conjugation among LAB has been indicated in previous chapters in discussions of conjugative transposons and antibiotic resistance plasmids as well as the association between conjugation and group II introns. Among the metabolic plasmids, the conjugative transfer of lactococcal lactose fermentation plasmids has been well reported  the sex factor causing a high frequency of recombination has been thoroughly analyze In addition to genes involved in the actual DNA transfer, the element contains a group II intron and a gene causing a “clumping” phenotype (cluA) associated with the high incidence of conjugation. The sex factor is also associated with an ISS1-type insertion element enabling its change of location from chromosome to plasmids. An important aspect of conjugation is the mobilization of normally nonconjugative plasmids by functional sex factors and conjugative genetic elements. For example, the enterococcal plasmid pAM b 1, which, as noted in Sec. III.E, can be conjugated to several species and genera of LAB, is able to efficiently induce the conjugative transfer of proteinase plasmids in lactococcal hosts.

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