5.4.1. Prokaryotic microorganisms

A variety of mechanisms are typical for transfer of DNA within prokaryotes and it can result in transfer of heritable properties.

These mechanisms of DNA transfer give to bacteria an advantage in response to environmental changes by adoption of new genetic information which could provide an efficient tool to sustain unfavorable selective pressure. Such kind of event is the wide spreading of antimicrobial resistance genes throughout microorganisms due to the introduction of antimicrobial agents in agriculture, healthcare, veterinary and medicine. A wide spread mechanism of gene transfer within prokaryotic systems is the conjugation, which is based on the presence of a plasmid in the donor cells or of conjugative transposons in the chromosome. The direct cell-to-cell contact helps these genetic elements to transfer copy(ies) of the plasmid or of the transposon(s) into the recipient cells. In bacteria a lot of plasmids have been identified and some of them lack the possibility for their own transfer. In this case it is facilitated by other plasmids.

The number of plasmids present within the bacterial cells could be different and this feature is common to bacterial populations, inhabiting different niches. These moving genetic elements - plasmids and transposons often can introduce new properties into the cells. An unique phenomenon in nature (as well as in experimental conditions) is the conjugative gene transfer from bacteria to eukaryotic cells (yeasts, filamentous fungi, animal and plant cells).

Another gene transfer process that is based on active uptake of extracellular DNA by bacteria into their cytoplasm is a natural transformation. This phenomenon was found to be characteristic of a limited number of bacteria occurring in major trophic and taxonomic groups. It was found that this process (transfer) could happen effectively during a specific growth phase of population growth called “competence”. The transformation can be accomplished by chromosomal DNA fragments or plasmids. This process can happen in specific physical or chemical conditions characterizing the phase of competence, when foreign DNA may enter bacterial cells. This type of transformation is often used in realization of gene technology.

The third type of gene transfer - transduction is also occurring in the microbial population and communities. Thus far it is mediated by bacterial viruses, which incidentally packed DNA of the last host cell, making it a donor one, and afterwards transmitted it to a recipient cell.

The specificity of the above described three mechanisms depends upon the genetic relatedness of the donor/receptor cells. The gene transfer could occur using these mechanisms within members of one species, but also between members of different species and genera. The so called “horizontal gene transfer” realized by these mechanisms is widely studied and admitted as very important for the genomic structure of bacterial species. The investigations of this phenomenon include also a whole genome sequence analysis.

Investigations in the field of natural gene transfer indicated that various transfer events could occur in natural habitats of bacteria including soil, rhizosphere, phyloplan, sediments, river epitops, foodstuffs, intestinal tract, mammalian oral cavity etc.

After the efficient transfer of foreign DNA into recipient cell it could be introduced into the genome via genomic integration (e.g. homologous recombination), or by formation of plasmid (in case of presence of replication origin). This process could be suspended by different reasons (like lack of nucleotide sequence homology or the presence of restriction endonucleases). It is evident that in case the novel genetic information gives an advantage for the recipient and allows its survival during changes in the natural ecosystem it tends to be preserved on the population level, when the selective pressure is durable. Thus, gene transfer could be considered as a phenomenon typical of the nature of prokaryotic microorganisms. It is a natural response to the changes in selective pressure of the environment, where the circulation of a gene or gene combination as well as the generation of several gene assemblies, give better opportunities in survival of microbial population.

Having in mind the natural character of gene transfer within bacterial community, which may ensure the wide spreading of recombinant constructs, it is preferable to use a chromosomal integration approach during the process of gene engineering manipulations. Inclusion of the genes into constructs bearing the target construct, which could give selective advantages under certain conditions, should also be avoided (e.g. antimicrobial resistance determinants). The procedure of elimination of each gene sequence, which is able to stimulate the random integration into other genomes, should be applied in the process of preparation of a desired construct.