A Dual-Replicon Shuttle Vector System for Heterologous Gene Expression in a Broad Range of Gram-Positive and Gram-Negative Bacteria

Abstract

Origin of replication (ori in theta-replicating plasmids or dso in rolling circle replicating plasmids) initiates plasmid replication in a broad range of bacteria. These two kinds of plasmids were both identified in Streptococcus, a genus composed of both human commensal bacteria and pathogens with the ability to cause severe community-acquired infections, including meningitides, septicemia, and respiratory tract diseases. Given the important roles of Streptococcus in the exchange of genetic elements with other symbiotic microbes, the genotypes and phenotypes of both Streptococcus spp. and other symbiotic species could be changed during colonization of the host. Therefore, an improved plasmid system is required to study the functional, complicated, and changeable genomes of Streptococcus. In this study, a dual-replicon shuttle vector system named pDRE was constructed to achieve heterologous gene expression. The vector system contained theta replicon for Escherichia coli. The origin of rolling circle replicon was synthesized according to pMV158 in Gram-positive bacteria. By measuring the products of inserted genes at multiple cloning sites, the ability of this vector system in the replication and expression of heterologous genes was assessed in four Streptococcus and three other Gram-positive bacteria: Bacillus subtilis, Lactococcus lactis, and Staphylococcus aureus. The results showed that the newly constructed vector could simultaneously replicate and express heterologous genes in a broad range of Gram-positive and Gram-negative bacteria, thus providing a potentially powerful genetic tool for further functional analysis.

Design and construction towards a pan-microbial toolkit

“…For ORIs that we were not able to construct, we did not determine the specific cause of failure. One possibility is that certain ORIs cannot be hosted on the same plasmid as the ColE1 ORI; however, the pCM62 ORI is derived from the RK2 ORI which was successfully created as a Level 0 part here and previous reports have described successful assembly of pVS1 and pLS1 ORIs with the ColE1 ORI on a single plasmid 16,29 . Finally, we created a non-functional "dummy ORI'' control part composed of a randomly-generated 48 bp DNA sequence.…”

Antimicrobial resistance. Characteristic of resistance to antimicrobial preparations of Gram-negative bacteria

“…Rezistența la preparatele antimicrobiene reprezintă o enormă criză globală în sănătatea publică și una dintre cele mai grave amenințări cu care se confruntă oamenii astăzi. Medicina modernă a determinat evoluția suplimentară a rezistenței antimicrobiene prin consumul excesiv de antibiotice [1][2][3].…”

“…Plus la aceasta, apar și alte întrebări. De curând a fost determinată incidența rezistenței antimicrobiene recent dobândite a bacteriilor aerobice Gam-negative potențial patogene [1,4].…”