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Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD- recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on targeted-antibacterial-plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step toward the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Drug-efflux by pump proteins is one of the major mechanisms of antibiotic resistance in bacteria. Here, we use quantitative fluorescence microscopy to investigate the real-time dynamics of drug accumulation and efflux in live E. coli cells. We visualize simultaneously the intrinsically fluorescent protein-synthesis inhibitor tetracycline (Tc) and the fluorescently labelled Tc-specific efflux pump, TetA. We show that Tc penetrates the cells within minutes and accumulates to stable intracellular concentration after ∼20 min. The final level of drug accumulation reflects the balance between Tc-uptake by the cells and Tc-efflux by pump proteins. In wild-type Tc-sensitive cells, drug accumulation is significantly limited by the activity of the multidrug efflux pump, AcrAB-TolC. Tc-resistance wild-type cells carrying a plasmid-borne Tn10 transposon contain variable amounts of TetA protein, produced under steady-state repression by the TetR repressor. TetA content heterogeneity determines the cells' initial ability to efflux Tc. Yet, efflux remains partial until the synthesis of additional TetA pumps allows for Tc-efflux activity to surpass Tc-uptake. Cells overproducing TetA no longer accumulate Tc and become resistant to high concentrations of the drug. This work uncovers the dynamic balance between drug entry, protein-synthesis inhibition, efflux-pump production, drug-efflux activity and drug-resistance levels., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The FtsK translocase pumps dsDNA directionally at ∼5 kb/s and facilitates chromosome unlinking by activating XerCD site-specific recombination at dif, located in the replication terminus of the Escherichia coli chromosome. We show directly that the γ regulatory subdomain of FtsK activates XerD catalytic activity to generate Holliday junction intermediates that can then be resolved by XerC. Furthermore, we demonstrate that γ can activate XerCD-dif recombination in the absence of the translocase domain, when it is fused to XerCD, or added in isolation. In these cases the recombination products are topologically complex and would impair chromosome unlinking. We propose that FtsK translocation and activation of unlinking are normally coupled, with the translocation being essential for ensuring that the products of recombination are topologically unlinked, an essential feature of the role of FtsK in chromosome segregation.