Discovery of Antagonist Peptides against Bacterial Helicase-Primase Interaction in B. stearothermophilus by Reverse Yeast Three-Hybrid

SummaryDeveloping small-molecule antagonists against protein-protein interactions will provide powerful tools for mechanistic/functional studies and the discovery of new antibacterials. We have developed a reverse yeast three-hybrid approach that allows high-throughput screening for antagonist peptides against essential protein-protein interactions. We have applied our methodology to the essential bacterial helicase-primase interaction in Bacillus stearothermophilus and isolated a unique antagonist peptide. This peptide binds to the primase, thus excluding the helicase and inhibiting an essential interaction in bacterial DNA replication. We provide proof of principle that our reverse yeast three-hybrid method is a powerful “one-step” screen tool for direct high-throughput antagonist peptide selection against any protein-protein interaction detectable by traditional yeast two-hybrid systems. Such peptides will provide useful “leads” for the development of new antibacterials

Untwisting of the DNA helix stimulates the endonuclease activity of Bacillus subtilis Nth at AP sites

Bacterial nucleoid associated proteins play a variety of roles in genome maintenance and dynamics. Their involvement in genome packaging, DNA replication and transcription are well documented but it is still unclear whether they play any specific roles in genome repair. We discovered that untwisting of the DNA double helix by bacterial non-specific DNA binding proteins stimulates the activity of a repair endonuclease of the Nth/MutY family involved in abasic site removal during base excision repair. The essential Bacillus subtilis primosomal gene dnaD, coding for a protein with DNA-untwisting activity, is in the same operon with nth and the promoter activity of this operon is transiently stimulated by H2O2. Consequently, dnaD mRNA levels persist high upon treatment with H2O2 compared to the reduced mRNA levels of the other essential primosomal genes dnaB and dnaI, suggesting that DnaD may play an important role in DNA repair in addition to its essential role in replication initiation. Homologous Nth repair endonucleases are found in nearly all organisms, including humans. Our data have wider implications for DNA repair as they suggest that genome associated proteins that alter the superhelicity of the DNA indirectly facilitate base excision repair mediated by repair endonucleases of the Nth/MutY family

Crystallization and X-ray diffraction analysis of the DNA-remodelling protein DnaD from Bacillus subtilis

Crystallization and preliminary X-ray diffraction analysis of the two domains of DnaD from B. subtilis is reported

DNA replication initiation in Bacillus subtilis: Structural and functional characterization of the essential DnaA-DnaD interaction

© 2018 The Author(s). The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation

Remodeling and control of homologous recombination by DNA helicases and translocases that target recombinases and synapsis

Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming "Displacement loops" (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork. Synapsis can be antagonized by limiting access of a recombinase to ssDNA and by dissociation of D-loops or heteroduplex formed by synapsis. Antagonists include DNA helicases and translocases that are identifiable in eukaryotes, bacteria and archaea, and which target synaptic and pre-synaptic DNA structures thereby controlling HR at early stages. Here we survey these events with emphasis on enabling DNA replication to be resumed from sites of blockage or collapse. We also note how knowledge of anti-recombination activities could be useful to improve efficiency of CRISPR-based genome editing

Evaluation of in-field efficacy of dietary ferric tyrosine on performance, intestinal health and meat quality of broiler chickens exposed to natural Campylobacter jejuni challenge

Campylobacter is an important pathogen commonly found in chickens that can cause severe acute gastroenteritis in humans. Despite intensive efforts to inhibit food-borne transmission of Campylobacter no effective strategy exists to reduce Campylobacter loads in farmed broilers. This study examined the capacity of a novel feed additive to lower Campylobacter jejuni populations and to improve growth efficiency of broiler chickens. A total of 384 male one-day-old broiler chicks were used in a 42-day trial. Birds were randomly allocated into four treatments with six replicates of sixteen chicks per pen. Three groups were fed the basal diets further supplemented with TYPLEX™ chelate (ferric tyrosine) at various concentrations (0.02, 0.05 and 0.20 g/kg, groups T2-T4, respectively). Control group (T1) was fed basal diets in mash form that did not contain added ferric tyrosine. Feed and water were provided ad libitum. At 20 days of age, broilers were exposed to natural C. jejuni challenge by introducing contaminated litter from a commercial farm. At day 25, pen litter samples analysed positive for C. jejuni, and the infection intensity was homogeneous among pens. At the end of the study C. jejuni counts in bird caeca were significantly reduced, by 2 log10 in the T4 group, compared to the T1 Control and T3 groups (p = 0.004). During this study, a natural infection with Eimeria tenella occurred at days 26-29. For animal welfare reasons all birds were treated with an anti-coccidial drug as recommended, for two consecutive days. At day 42, diarrhoea was observed on the litter in only 1 of 6 pens in the T4 group, but in 5 of 6 pens in the T1 Control group. In addition, autopsies showed that the T4 group had the highest percentage of birds with normal intestinal tracts. The T1 group had the lowest percentage of birds with infection-free tracts, and higher incidence of coccidiosis and bloody diarrhoea. At 42 days of age all birds were slaughtered and samples collected for further analysis. Birds in the T4 group tended to exhibit improved weight gain and feed efficiency, a result that warrants further investigation. Collectively, our data suggest that addition of ferric tyrosine at 0.20 g/kg exerts a protective effect against C. jejuni and coccidiosis

Ferric Quinate (QPLEX) interacts with the Major Outer Membrane Protein (MOMP) of Campylobacter jejuni and enters through the porin channel into the periplasmic space

Ferric chelates like ferric tyrosinate (TYPLEX) and the closely related ferric quinate (QPLEX) are structural mimics of bacterial siderophores. TYPLEX has been trialled as a feed additive in farming of commercial broilers, reducing Campylobacter loads by 2–3 log10 and leading to faster growth and better feed consumption. These ferric chelates offer a good alternative feed additive to antibiotics helping to reduce the indiscriminate use of preventative antibiotics in broiler farming to control Campylobacter infections. In this study, we show that QPLEX binds to the Major Outer Membrane Protein (MOMP) of C. jejuni NCTC11168. MOMP is an essential and abundant outer membrane porin on the surface of the bacteria, acting as an adhesin to help establish infection by mediating attachment of C. jejuni onto the gut epithelium of broilers and establish infection. Using carbene footprinting, we map the MOMP-QPLEX interaction and show by complementary in silico docking that QPLEX enters the porin channel through interactions at the extracellular face, translocates down the channel through a dipole transverse electric field towards the opposite end and is released into the periplasm at the intracellular face of MOMP. Our studies suggest a potential mechanism for the non-antibiotic anti-Campylobacter activity of these ferric chelates

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a “ratchet” for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by “reeling” ssDNA

Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR

The Escherichia coli MelR protein is a transcription activator that autoregulates its own promoter by repressing transcription initiation. Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites. In this work, we have investigated the different determinants needed for optimal repression and their spatial requirements. We show that repression requires a complex involving four DNA-bound MelR molecules, and that the global CRP regulator plays little or no role

Defining the intrinsically disordered C-terminal domain of SSB reveals DNA-mediated compaction

The bacterial single-stranded DNA (ssDNA) binding protein SSB is a strictly conserved and essential protein involved in diverse functions of DNA metabolism, including replication and repair. SSB comprises a well-characterized tetrameric core of N-terminal oligonucleotide binding OB folds that bind ssDNA and four intrinsically disordered C-terminal domains of unknown structure that interact with partner proteins. The generally accepted, albeit speculative, mechanistic model in the field postulates that binding of ssDNA to the OB core induces the flexible, undefined C-terminal arms to expand outwards encouraging functional interactions with partner proteins. In this structural study, we show that the opposite is true. Combined small-angle scattering with X-rays and neutrons coupled to coarse-grained modeling reveal that the intrinsically disordered C-terminal arms are relatively collapsed around the tetrameric OB core and collapse further upon ssDNA binding. This implies a mechanism of action, in which the disordered C-terminal domain collapse traps the ssDNA and pulls functional partners onto the ssDNA