Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylSZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNApyl, such as pylSZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated.

Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylSZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNApyl, such as pylSZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated.

Function Characterization of Endogenous Plasmids in Cronobacter sakazakii and Identification of p-Coumaric Acid as Plasmid-Curing Agent

Virulence traits and antibiotic resistance are frequently provided by genes located on plasmids. However, experimental verification of the functions of these genes is often lacking due to a lack of related experimental technology. In the present study, an integrated suicide vector was used to efficiently and specifically delete a bacterial endogenous plasmid in Cronobacter sakazakii. The pESA3 plasmid was removed from C. sakazakii BAA-894, and we confirmed that this plasmid contributes to the invasion and virulence of this strain. In addition, the pGW1 plasmid was expunged from C. sakazakii GZcsf-1, and we confirmed that this plasmid confers multidrug resistance. We further screened plasmid-curing agents and found that p-coumaric acid had a remarkable effect on the curing of pESA3 and pGW1 at sub-inhibitory concentrations. Our study investigated the contribution of endogenous plasmids pESA3 and pGW1 by constructing plasmid-cured strains using suicide vectors and suggested that p-coumaric acid can be a safe and effective plasmid-curing agent for C. sakazakii.

Repurposing CRISPR-Cas Systems as Genetic Tools for the Enterobacteriales

Over the last decade, the study of CRISPR-Cas systems has progressed from a newly discovered bacterial defense mechanism to a diverse suite of genetic tools that have been applied across all domains of life. While the initial applications of CRISPR-Cas technology fulfilled a need to more precisely edit eukaryotic genomes, creative “repurposing” of this adaptive immune system has led to new approaches for genetic analysis of microorganisms, including improved gene editing, conditional gene regulation, plasmid curing and manipulation, and other novel uses.

Plasmid-Curing, Antimicrobial, Antioxidant Properties and Phytochemical Analysis of Medicinal Plants from North East India

The medicinal importance of plants is in their bioactive substances which exert definite physiological action on the human body. In the present investigation antimicrobial activities of Garcinia pedunculata, Phlogacanthus thyrsiformis, and Ziziphus mauritiana were studied against microbial strains using agar disc diffusion. In-vitro phytochemical screening for chloroform, isoamyl alcohol and water extracts of parts of plants was performed. For MIC (minimum inhibitory concentration), grid method was used. The antioxidant activity of the plant extracts was studied using the ferric reducing antioxidant method and bioautography was studied using Thin Layer Chromatography (TLC). Garcinia pedunculata and Phlogacanthus thyrsiformis extracts showed highest antimicrobial activity. In-vitro phytochemical screening for chloroform, isoamyl alcohol and water extracts of parts of plants showed positive results for alkaloids, saponins, steroids, triterpenes, flavonoids and diterpenes. The MIC value of Garcinia pedunculata, Phlogacanthus thyrsiformis and Ziziphus mauritiana was 2560, 1280 and 2560 µL, respectively. The antioxidant activity revealed that there was an increase in absorbance with the increase of sample concentration. In Thin Layer Chromatography-Bioautography, chloroform extract of Garcinia pedunculata and Ziziphus mauritiana showed activity with zones of inhibition on bioautograms. The chloroform extract of Ziziphus mauritiana was found to be effective with curing efficiency for E. coli K12 (RP4), E. coli (pBR322) and E. coli (pRK2013) 62%, 57% and 49% respectively. Petroleum ether extract of Phlogacanthus thyrsiformis cured E. coli K12 (RP4), E. coli (pBR322) and E. coli (pRK2013) at 38%, 42% and 35% curing efficiencies respectively. This is the first report of plasmid curing by using chloroform Ziziphus mauritiana and petroleum ether extract of Phlogacanthus thyrsiformis. The present investigation has revealed applications and significance of plant extracts of Garcinia pedunculata, Phlogacanthus thyrsiformis, and Ziziphus mauritiana as plasmid curing, antimicrobial and antioxidant agents to control infections and spread of antibiotic resistance in pathogenic bacteria.