Plasmids and temperate phages influence each other’s transfer rates

Accessory genes, such as antibiotic resistance genes (ARGs) can spread horizontally by mobile genetic elements, including plasmids and (temperate) bacteriophages leading to increased bacterial fitness in particular environments. In contrast to plasmids, temperate phages i.e. viruses that can incorporate their own genetic material into their host bacteria (then called lysogen) can additionally increase their hosts’ fitness through their ability to kill phage-susceptible competitors. However, in contrast to ARG-transfer by plasmids (conjugation), which has been extensively studied, ARG-transfer by phages (lysogenization) has received far less attention. Here we combined experiments using E. coli, phage lambda and the Rp4 plasmid (phage and plasmid both with an ampicillin resistance gene) and mathematical models to test which mechanism (lysogenization or conjugation) dominates under which conditions. In the absence of selection and in two-species experiments (Donor & Recipient), conjugation was significantly more common than lysogenization. However, in three-species experiments (DonorPlasmid, DonorPhage& Recipient) lysogenization rates increased by several orders of magnitude. By using phage-friendly environments that favour phage adsorption we were able to shift the ratio between lysogens and transconjugants even further towards lysogens and in extreme-events to the extinction of transconjugants. Mathematical models additionally allowed us to investigate how starting population sizes of both donors and the recipient influence conjugation and lysogenization dynamics. Taken together, our results suggest that plasmids and temperate phages can influence each other’s transfer rates which, in the present study system, seems to be largely driven by population size effects.

Directed evolution of a transcription factor PbrR to improve lead selectivity and reduce zinc interference through dual selection

Directed evolution has been proven as a powerful tool for developing proteins and strains with novel or enhanced features. In this study, a dual selection system was designed to tune the binding specificity of a transcription factor to a particular ligand with the ampicillin resistance gene amp (ON selection) as the positive selection marker and the levansucrase gene sacB (OFF selection) as the negative selection marker. It was applied to the lead responsive transcription factor PbrR in a whole-cell lead biosensor previously constructed in our lab. After multiple rounds of ON-OFF selection, two mutants with higher specificity for lead were selected. Structural analysis revealed that the mutation C134 located on the metal-binding loop at the C-terminal of PbrR is likely associated with the enhanced binding to both lead and cadmium. The double mutations D64A and L68S close to the metal-binding residue C79 may lead to the reduced binding specificity toward zinc ions. This dual selection system can be applied to engineer the specificity of other transcription factors and provide fine-tuned tools to synthetic biology.

Elimination of transforming activity and gene degradation during UV and UV/H2O2 treatment of plasmid-encoded antibiotic resistance genes

This study determined deactivation of transforming activity of an ARG and the ARG degradation during UV and UV/H2O2 treatment of plasmid pUC19 containing an ampicillin resistance gene.

The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes.

Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves

Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P. dioica plants, produces both free 3′-OH and 5′-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [α-32P]dCTP and [γ-32P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E. coli HB101 competent cells in comparison to untreated control plasmid DNA.