DNA Sampling: a method for probing protein binding at specific loci on bacterial chromosomes

We describe a protocol, DNA sampling, for the rapid isolation of specific segments of DNA, together with bound proteins, from Escherichia coli K-12. The DNA to be sampled is generated as a discrete fragment within cells by the yeast I-SceI meganuclease, and is purified using FLAG-tagged LacI repressor and beads carrying anti-FLAG antibody. We illustrate the method by investigating the proteins bound to the colicin K gene regulatory region, either before or after induction of the colicin K gene promoter

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations

Unique relationships between phages and endospore-forming hosts

As part of their survival strategy under harsh environmental conditions, endospore-forming bacteria can trigger a sporulation developmental program. Although the regulatory cascades that precisely control the transformation of vegetative bacteria into mother cells and resilient spores have been described in detail, less is known about how bacteriophages that prey on endospore-formers exploit sporulation. Herein, we argue that phages infecting these bacteria have evolved several specific molecular mechanisms, not yet known in other bacteria, that manifest from the phage-driven alliance to negative effects on the host. We anticipate that the relationships between phages and endospore-formers outlined here will inspire studies on phage ecology and evolution, and could facilitate important advances in the development of phage therapies against pathogenic spore-formers

Surface plasmon resonance approach to study drug interactions with SARS-CoV-2 RNA-dependent RNA polymerase highlights treatment potential of suramin

The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is essential for virus replication, therefore it is a promising drug target. Here we present a surface plasmon resonance approach to study the interaction of RdRp with drugs in real time. We monitored the effect of favipiravir, ribavirin, sofosbuvir triphosphate PSI-7409 and suramin on RdRp binding to RNA immobilized on the chip. Suramin precluded interaction of RdRp with RNA and even displaced RdRp from RNA

Sublethal Concentrations of Ciprofloxacin Induce Bacteriocin Synthesis in Escherichia coli

Antibiotics that interfere with DNA replication, as well as cell wall synthesis, induce the SOS response. In this report, we show that ciprofloxacin induces synthesis of colicins, narrow-spectrum antibiotics frequently produced by Escherichia coli strains, in an SOS-dependent manner

Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage

The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription.Finnish Centre of Excellence in Biological Interactions [252411]; (cofunded under the Marie Curie Actions of the European Commission) [FP7-COFUND];EMBO[ASTF 286-2013 to N.F.]; Academy of Finland [251106 to J.K.B.]; Slovenian Research Agency [P1-0207 to M.B. and I0-0021 to V.H. and G.A.]; Spanish Ministry of Economy and Competitiveness [BFU2011-23645 to M.S.]. Funding for open access charge: Academy of Finland.Peer Reviewe