Propagation of Recombinant Genes through Complex Microbiomes with Synthetic Mini-RP4 Plasmid Vectors

Abstract

The promiscuous conjugation machinery of the Gram-negative plasmid RP4 has been reassembled in a minimized, highly transmissible vector for propagating genetically encoded traits through diverse types of naturally occurring microbial communities. To this end, the whole of the RP4-encoded transfer determinants (tra, mob genes, and origin of transfer oriT) was excised from their natural context, minimized, and recreated in the form of a streamlined DNA segment borne by an autoselective replicon. The resulting constructs (the pMATING series) could be self-transferred through a variety of prokaryotic and eukaryotic recipients employing such a rationally designed conjugal delivery device. Insertion of GFP reporter into pMATING exposed the value of this genetic tool for delivering heterologous genes to both specific mating partners and complex consortia (e.g., plant/soil rhizosphere). The results accredited the effective and functional transfer of the recombinant plasmids to a diversity of hosts. Yet the inspection of factors that limit interspecies DNA transfer in such scenarios uncovered type VI secretion systems as one of the factual barriers that check otherwise high conjugal frequencies of tested RP4 derivatives. We argue that the hereby presented programming of hyperpromiscuous gene transfer can become a phenomenal asset for the propagation of beneficial traits through various scales of the environmental microbiome.

Taking Synthetic Biology to the Seas: From Blue Chassis Organisms to Marine Aquaforming

“…Utilizing a minimized conjugation machinery, the effectiveness of these constructs was validated by transferring a gene encoding for green fluorescent protein from and to numerous donor and recipient strains, and notably even to consortia in the soil microbiome. There are tailored versions of this genetic tool for both gram‐negative and gram‐positive bacteria, as well as for eukaryotes [104] . If this system is shown to be efficient in the transmission of larger gene clusters as well, it might be a promising starting point to propagate pathways for bioremediation or greenhouse gas fixation through marine microbiomes.…”

“…There are tailored versions of this genetic tool for both gram-negative and gram-positive bacteria, as well as for eukaryotes. [104] If this system is shown to be efficient in the transmission of larger gene clusters as well, it might be a promising starting point to propagate pathways for bioremediation or greenhouse gas fixation through marine microbiomes. The deployment of self-propagating biologicals could be realized by using aerosols or wind-carried particles to reach a sufficient initial spread in a marine habitat.…”

SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes

“…Still within the realm of fully-standardized plasmids it is worth to highlight pSEVA2a2d1, which bear a double origin of replication ( 24 ) that enables constructs to become established in Gram-positive hosts ( 25 ). This adds to the already existing branch of SEVA vectors that replicate both in Gram-negative bacteria and actinomycetes ( 26 ).…”