Metrics details Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB–KorA system in Escherichia coli we show that long-range gene silencing on the plasmid RK2 a source of multi-drug resistance across diverse Gram-negative bacteria is achieved cooperatively by a DNA-sliding clamp We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away We resolved the tripartite crystal structure of a KorB–KorA–DNA co-complex revealing that KorA latches KorB into a closed clamp state DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor and provide an alternative mechanism for long-range regulation of gene expression in bacteria we investigate KorAB–CTP interaction and provide a unifying model for KorAB-mediated long-range gene silencing We find that KorB binds CTP to form a protein clamp that can entrap and slide along DNA to mediate long-range transcriptional repression likely by allowing KorB to reach target promoters from distal OB sites finding that the DNA-binding KorA is also a clamp-locking protein that docks underneath the DNA-binding domain (DBD) of KorB to latch the closed-clamp state Our data suggest that the KorA–KorB interaction stalls KorB sliding at target core promoter elements and exploits an inherently unstable open RNA polymerase (RNAP)–promoter complex to exclude RNAP holoenzymes from the promoters KorA–KorB interaction also increases the residence time of KorAB on DNA we demonstrate how a DNA-binding and clamp-locking protein KorA allows KorB to switch functions between sliding and stalling on DNA to act as an effective repressor provide unanticipated insights into long-range transcriptional repression mechanisms in bacteria and thus N146 was also selected for mutagenesis in this study NTP hydrolysis rates of KorB (WT and variants) were measured at increasing concentrations of NTP Source data our data show that KorB is an OB DNA-stimulated CTPase Source data We interpret these events as either new KorB proteins being loaded at 8×OB or existing KorB proteins diffusing and occasionally re-binding to OB when they re-encounter this cluster indicating that KorB–CTP does not condense DNA under the tested conditions we observed unspecific condensation at high concentrations (1–2 µM) BsParB was not able to condense at a lower concentration (500 nM) because BsParB does not recognize OB sites on our DNA substrate our data suggest that the ability of KorB to bind CTP and close the clamp to slide on DNA is crucial for long-range gene silencing Source data showing that KorB∆N30∆CTD in the tripartite complex has already adopted an NTD-engaged conformation even though CTPɣS was not added to the crystallization set-up (see Supplementary Information for further structural analysis and discussion) we reasoned that KorA–DNA may facilitate or capture KorB∆N30∆CTD in the NTD-engaged state directly interacts with the DBD of KorB to lock KorB in an NTD-engaged closed-clamp state Source data KorB localized between the OA and OB sites Case I is most frequent at ~45% of the 182 recorded events and consistent with OA-bound KorA and/or OA-bound KorA–KorB complex blocking the sliding of KorB–CTP We occasionally observed trespassing of KorB beyond the OA site; trespassing is likely due to a transient disruption of the KorA–KorB complex or KorB diffusing beyond OA before the complex formation KorB localized between OA and the OB-proximal bead We interpret case II as follows: KorB was bound to OB and a subsequent KorB protein binding to OB might act as a roadblock preventing a previously loaded KorB from passing through the OB site thereby restricting KorB to either side of OB The higher occurrence of case I compared with case II is likely due to the higher chance of KorB finding KorA within the OA–OB region given a particular lifetime of the KorB–DNA interaction there were stable co-localizations of both KorA and KorB at OA Case III likely reflects a single diffusing KorB protein binding to KorA we never observed KorB localization at OA without KorA we observed mostly static KorA and KorB binding at OA and OB these results suggest that OA-bound KorA captures a DNA-entrapped sliding clamp of KorB to cooperatively repress promoters Promoter scaffold from the RK2 korABF operon (PkorA) is shown with core promoter elements (underlined) Differences to the PkorA:λPR discriminator are depicted coli Eσ70 holoenzyme assembled on 100 bp PkorA DNA (Eσ70:DNA) with and without 2.5-fold excess KorA dimer in 150 mM ammonium acetate pH 7.5 and 0.01% Tween-20 Deconvolved nMS spectra of Eσ70:DNA with and without 2.5-fold excess KorB dimer in 500 mM ammonium acetate pH 7.5 and 0.01% Tween-20 Top: representative gel close-up on the abortive RNA product (5′-ApUpG-3′) transcribed by Eσ70 in in vitro abortive initiation half-life assays on the two PkorA linear scaffold variants Bottom: plot of fraction of competitor-resistant open complexes (from normalized abortive RNA band intensities) against time Data points from three experimental replicates are mean values ± s.e.m Some error bars are too small or lead to negative values and thus were omitted Estimated half-lives are shown adjacent to exponential decay trend lines WT PkorA and PkorA:λPR discriminator in vitro transcription repression of Eσ70 in the presence of fivefold excess KorA and/or KorB Data points from at least three experimental replicates are normalized to holoenzyme only control as mean values ± s.e.m Eσ70 + KorA and Eσ70 + KorA + KorB conditions on WT PkorA has n = 4 while the rest are n = 3; this remains valid as repression quantities are normalized to a Eσ70-only control and background-corrected for each gel run P values were calculated by unpaired two-tailed Welch’s t-tests; *P ≤ 0.05 The P values are 3.66 × 10−3 (Eσ70 + KorA) 0.635 × 10−3 (Eσ70 + KorB) and 31.5 × 10−3 (Eσ70 + KorA + KorB) Source data coupled with the observations of KorA and KorB binding to DNA but not to Eσ70 resulting in frequent RNAP dissociation that allows KorA and KorB to bind their respective operator sites and form a repressome on the promoter that occludes RNAP from the promoter Our findings suggest that KorAB exploits RK2 promoter kinetic instabilities (that is weak discriminators leading to short half-lives) to competitively occlude RNAPs from DNA The presence of CTP (orange) and OB DNA likely triggers KorB clamp closing KorB can slide away from the OB site by diffusion while entrapping DNA CTP hydrolysis and/or the release of hydrolytic products (CDP and inorganic phosphate Pi) likely reopen the clamp to release DNA Substitutions that affect various KorB functions are also indicated on the schematic diagram KorA (magenta) bound at OA can form a complex with and promote or trap KorB in a closed clamp state The tripartite KorAB–DNA reduces the release of KorB from DNA as well as the release of KorA from OA DNA A model for KorA–KorB cooperation to enhance long-range transcription repression OB can position kilobases away from the target core promoter elements while OA is almost invariably near these core promoter elements Owing to an unfavourable discriminator sequence the RNAP holoenzyme–promoter DNA complex is inherently unstable thus only providing an unstable steric hindrance to occlude RNAP (magenta) from the core promoter elements resulting in weak transcriptional repression closes the clamp and slides by diffusion to reach the distal OA site OA-bound KorA captures and locks KorB in a clamp-closed conformation the KorAB–DNA co-complex presents a larger and more stable steric hindrance the KorAB–DNA co-complex can exploit the unstable RPo and occludes RNAP more effectively hence stronger transcriptional repression than each protein alone can provide Our insights into KorAB here might have an impact beyond the bacterial transcription field providing a conceptual advance in our understanding of phage bacterial and eukaryotic transcriptional regulation coli strains were grown in lysogeny broth (LB) medium the media was supplemented with antibiotics at the following concentrations (liquid/solid (μg ml−1)): carbenicillin (50/100) streptomycin (50/50) and tetracycline (12.5/12.5) Plasmids and strains used or generated in this study are listed in Supplementary Tables 1 and 2 a double-stranded DNA fragment containing a desired sequence was chemically synthesized (gBlocks The target plasmid was double-digested with restriction enzymes and gel-purified A 10 μl reaction mixture was created with 5 μl 2× Gibson master mix (NEB) and 5 μl of combined equimolar concentration of purified backbone and gBlock(s) This mixture was incubated at 50°C for 60 min Gibson assembly was possible owing to shared sequence similarity between the digested backbone and the gBlock fragment(s) All resulting plasmids were verified by Sanger sequencing (Genewiz) or whole-plasmid sequencing (Plasmidsaurus) DNA fragments containing mutated korB genes (korB*) were chemically synthesized (gBlocks The NdeI-HindIII-cut pET21b plasmid backbone and korB* gBlocks fragments were assembled using a 2× Gibson master mix (NEB) Gibson assembly was possible owing to a 37 bp sequence shared between the NdeI-HindIII-cut pET21b backbone and the gBlocks fragment DNA fragments containing mutated korA genes (korA*) were chemically synthesized (gBlocks The NdeI-HindIII-cut pET21b plasmid backbone and korA* gBlocks fragments were assembled using a 2× Gibson master mix (NEB) The SacI-HindIII-cut pBAD33 plasmid backbone and korB* gBlocks fragments were assembled using a 2× Gibson master mix (NEB) Gibson assembly was possible owing to a 38 bp sequence shared between the SacI-HindIII-cut pBAD33 backbone and the gBlocks fragment The EcoRI-SalI-cut pDM2.1 plasmid backbone and korA* gBlocks fragments were assembled using a 2× Gibson master mix (NEB) Gibson assembly was possible owing to a 38 bp sequence shared between the EcoRI-SalI-cut pDM2.1 backbone and the gBlocks fragment DNA fragments containing mutated PkorA promoters (PkorA*) were chemically synthesized (gBlocks The BamHI-cut pSC101 plasmid backbone and PkorA* gBlocks fragments were assembled using a 2× Gibson master mix (NEB) Gibson assembly was possible owing to a 38 bp sequence shared between the BamHI-cut pSC101 backbone and the gBlocks fragment A 146 bp DNA fragment containing PkorA was chemically synthesized (gBlocks IDT) and subsequently 5′-phosphorylated using T4 PNK (NEB) Phosphorylated 146 bp PkorA DNA was blunt-end ligated with a dephosphorylated SmaI-cut pUC19 using T4 DNA ligase (NEB) To clone the 146 bp PkorA:λPR discriminator DNA into pUC19 pUC19::146bp-PkorA was used as a template for site-directed mutagenesis with Q5 DNA Polymerase (NEB) using the primers 5′-AACATTTCTCGCACG-3′ and 5′-TAGCTAAACTGGTTGCATGTGCTGGCG-3′ at an annealing temperature of 57 °C The resulting PCR product was introduced into E and carbenicillin-resistant colonies were isolated plasmids were isolated and verified by whole-plasmid sequencing (Plasmidsaurus) Plasmids were introduced/co-introduced into E. coli DH5α or E. coli BL21 pLysS via heat shock transformation (42 °C, 30 s) in the required combinations (Supplementary Table 2) Proteins used or generated in this study are listed in Supplementary Table 3 C-terminally His-tagged KorA and KorB (WT and mutants) were expressed from the plasmid pET21b in E coli Rosetta (BL21 DE3) pLysS competent cells (Merck) Overnight culture (120 ml) was used to inoculate 6 l of LB with selective antibiotics Cultures were incubated at 37 °C with shaking at 220 r.p.m Cultures were cooled for 2 h at 4 °C before isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM The cultures were incubated overnight at 16 °C with shaking at 220 r.p.m before cells were pelleted by centrifugation Cell pellets were resuspended in buffer A (100 mM Tris–HCl pH 8.0) with 5 mg lysozyme (Merck) and a cOmplete EDTA-free protease inhibitor cocktail tablet (Merck) at room temperature for 30 min with gentle rotation Cells were lysed on ice with 10 cycles of sonication: 15 s on/15 s off at an amplitude of 20 µm and pelleted at 32,000 g for 35 min at 4 °C and the supernatant filtered through a 0.22 µm sterile filter (Sartorius) Clarified lysate was loaded onto a 1 ml HisTrap HP column (Cytiva) pre-equilibrated with buffer A Protein was eluted from the column using an increasing gradient of imidazole (10–500 mM) in the same buffer Desired protein fractions were pooled and diluted in buffer B (100 mM Tris–HCl pH 8.0) until the final concentration of NaCl was 60 mM Pooled fractions were loaded onto a 1 ml Heparin HP column (Cytiva) pre-equilibrated with buffer B Protein was eluted from the column using an increasing gradient of NaCl (20–1,000 mM) in the same buffer Desired protein fractions were pooled and loaded onto a preparative-grade HiLoad 16/600 Superdex 75 pg gel filtration column (GE Healthcare) pre-equilibrated with elution buffer (10 mM Tris–HCl Desired fractions were identified and analysed for purity via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) before being pooled Aliquots were flash frozen in liquid nitrogen and stored at −80 °C For protein samples to be used for protein–nucleotide binding ITC experiments Mg2+ was introduced via an overnight dialysis step at 4 °C in buffer containing 10 mM Tris–HCl pH 8.0 before concentration and quantification as described above Protein preparations for BMOE crosslinking were purified using a one-step Ni-affinity column with all buffers adjusted to pH 7.4 for optimal crosslinking Purified proteins were subsequently desalted using a PD-10 column (Merck) before being concentrated using an Amicon Ultra-4 10 kDa cut-off spin column (Merck) Final protein samples were aliquoted and stored at −80 °C in storage buffer (100 mM Tris–HCl Both biological (new sample preparations from a stock aliquot) and technical (same sample preparation) replicates were performed for assays in this study Protein concentrations were determined by Bradford assay and reported as concentrations of KorA or KorB dimers ω) as well as β′-PPX-His10 (PPX; PreScission protease site GE Healthcare) were co-introduced with a pACYCDuet-1::E.coli rpoZ into E coli BL21(DE3) to ensure saturation of all RNAPs with E Cells were grown in the presence of 100 µg ml−1 ampicillin and 34 μg ml−1 chloramphenicol to an OD600 of ~0.6 in a 37 °C shaker Protein expression was induced with 1 mM IPTG (final concentration) for 4 h at 30 °C Cells were collected by centrifugation and resuspended in 50 mM Tris–HCl pH 8.0 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail For 200× protease inhibitor cocktail (40 ml volume) the following are dissolved into 100% ethanol: 696 mg PMSF After lysis by French press (Avestin) at 4 °C the lysate was centrifuged twice for 30 min each Thermo Fisher Scientific) was slowly added to the supernatant to a final concentration of ~0.6% PEI with continuous stirring The mixture was stirred at 4 °C for an additional 25 min The pellets were washed three times with 50 mM Tris–HCl pH 8.0 the pellets were homogenized and then centrifuged again RNAP was eluted by washing the pellets three times with 50 mM Tris–HCl pH 8.0 1× protease inhibitor cocktail and 1 mM PMSF The PEI elutions were combined and precipitated overnight with ammonium sulfate at a final concentration of 35% w/v and the pellets were resuspended in 20 mM Tris–HCl pH 8.0 5% w/v glycerol and 1 mM β-mercaptoethanol (BME) The mixture was loaded onto two 5 ml HiTrap IMAC HP columns (Cytiva) for a total column volume of 10 ml RNAP(β′-PPX-His10) was eluted at 250 mM imidazole in column buffer (20 mM Tris–HCl pH 8.0 The eluted RNAP fractions were combined and dialysed into 10 mM Tris–HCl pH 8.0 The sample was then loaded onto a 40 ml Bio-Rex-70 column (Bio-Rad) 5% w/v glycerol and 5 mM DTT in isocratic steps of increasing concentration of NaCl (eluted at 0.5 M NaCl) then loaded onto a 320 ml HiLoad 26/600 Superdex 200 column (Cytiva) pre-equilibrated in gel filtration buffer (10 mM Tris–HCl pH 8.0 The eluted RNAP was concentrated to ~8–10 mg ml−1 by centrifugal concentration flash frozen in liquid nitrogen and stored at −80 °C Plasmid pSAD1403 (ref. 61) was introduced into E The cells were grown in the presence of 50 μg ml−1 kanamycin to an OD600 of ~0.6 at 37 °C Protein expression was induced with 1 mM IPTG for 1–1.5 h at 30 °C Cells were collected by centrifugation and resuspended in 20 mM Tris–HCl pH 8.0 1 mM PMSF and 1× protease inhibitor cocktail cell debris was removed by centrifugation twice The lysate was loaded onto two 5 ml HiTrap IMAC HP columns (Cytiva) for a total column volume of 10 ml His10-SUMO-σ70 was eluted at 250 mM imidazole in 20 mM Tris–HCl pH 8.0 cleaved with Ulp1 protease and dialysed against 20 mM Tris–HCl pH 8.0 resulting in a final concentration of 25 mM imidazole The cleaved sample was loaded onto one 5 ml HiTrap IMAC HP to remove His10-SUMO tag along with any remaining uncleaved σ70 Untagged σ70 fractions were pooled and diluted with 10 mM Tris–HCl pH 8.0 5% w/v glycerol and 1 mM DTT until the conductivity corresponds to NaCl concentration slightly below 200 mM The diluted sample was injected onto three 5 ml HiTrap Heparin HP columns (total column volume of 15 ml; Cytiva) which were pre-equilibrated at the same diluent buffer but with 200 mM NaCl with the first major peak as the target peak The target peak sample was pooled and concentrated by centrifugal filtration before being loaded onto a HiLoad 16/60 Superdex 200 (Cytiva) which was pre-equilibrated in 20 mM Tris–HCl pH 8.0 supplemented with glycerol to a final concentration of 20% w/v Crystallization screens for both KorBΔN30ΔCTD–CTPγS and KorBΔN30ΔCTD–KorA–OA complexes were performed in sitting-drop vapour diffusion format in MRC2 96-well crystallization plates Drops consisted of 0.3 μl precipitant solution and 0.3 μl protein complex with incubation at 293 K Purified His-tagged KorBΔN30ΔCTD was premixed at 20 mg ml−1 with 1 mM MgCl2 and 1 mM CTPγS in buffer containing 10 mM Tris–HCl and 150 mM NaCl The KorBΔN30ΔCTD–CTPγS crystals grew in a solution containing 160 mM LiOAc and 2.0 M ammonium sulfate Suitable crystals were cryoprotected with 20% (v/v) ethylene glycol and mounted in Litholoops (Molecular Dimensions) Crystals were flash-cooled by plunging into liquid nitrogen Purified His-tagged KorBΔN30ΔCTD was combined with purified His-tagged KorA in equimolar concentrations before being purified by gel filtration as described above The protein complex was premixed at 20 mg ml−1 with a 14 bp dsDNA (OA TGTTTAGCTAAACA) at a molar ratio 1:1.2 (protein complex to DNA) in buffer containing 10 mM Tris–HCl and 150 mM NaCl Crystals grew in a solution containing 1.95 M ammonium sulfate and 0.1 M NaOAc Suitable crystals were cryoprotected with 25% (v/v) glycerol and mounted in Litholoops (Molecular Dimensions) X-ray data for KorBΔN30ΔCTD–CTPγS were collected from a single crystal at a wavelength of 0.9179 Å and processed to 2.3 Å resolution in space group P212121 with approximate cell parameters of a = 58.7 Analysis of the likely composition of the asymmetric unit suggested that it could contain between four and eight copies of the KorB subunit with an estimated solvent content in the range of 43–72% and the predicted local distance difference test (pLDDT) scores were generally good (for example the predicted aligned error scores indicated a two-domain structure with very low confidence in the relative placement of the two domains the predicted aligned error scores suggested high confidence in the relative placement of all four domains all five independently generated models were closely superposable X-ray data for KorBΔN30ΔCTD–KorA–OA were collected from a single crystal at a wavelength of 0.9763 Å (2 × 360° passes) and processed to 2.7 Å resolution in space group C2 with approximate cell parameters of a = 173.2 Analysis of the likely composition of the asymmetric unit suggested that it contained a single complex comprising two copies of each of the KorA and KorB subunits and a single DNA duplex giving an estimated solvent content of 61% Palindromic single-stranded DNA oligonucleotides (OB TGTTTAGCTAAACA) (100 µM in 1 mM Tris–HCl pH 8.0 5 mM NaCl buffer) were heated at 98 °C for 5 min before being left to cool down to room temperature overnight to form 50 µM dsDNA The core sequence of OB or OA is underlined CTP hydrolysis was monitored using an EnzCheck Phosphate Assay Kit (Thermo Fisher Scientific) Samples (100 µl) containing a reaction buffer supplemented with an increasing concentration of CTP (1 and 1 µM dimer concentration of KorB (WT or mutants) were assayed in a CLARIOstar Plus plate reader (BMG Labtech) at 25 °C for 5 h with readings every 2 min with continuous orbital shaking at 300 r.p.m The reaction buffer (1 ml) typically contained 740 μl ultrapure water 50 μl 20× reaction buffer (100 mM Tris–HCl 200 μl 2-amino-6-mercapto-7-methylpurine riboside (MESG) substrate solution and 10 μl purine nucleoside phosphorylase enzyme (one unit) Reactions with buffer only or buffer + CTP + 24 bp OB DNA only were also included as controls The inorganic phosphate standard curve was also constructed according to the instruction guidelines The CTPase rates were calculated using a linear regression fitting in GraphPad Prism 9 Error bars represent standard deviations from triplicate experiments A 50 µl mixture of 8 µM dimer concentration of KorB WT or mutants ± 1 mM NTP ± 0.5 µM 24 bp dsDNA containing OB or scrambled OB was assembled in a reaction buffer (10 mM Tris–HCl pH 7.4 100 mM NaCl and 1 mM MgCl2) and incubated for 5 min at room temperature BMOE was added to a final concentration of 1 mM and the reaction was quickly mixed by three pulses of vortexing The reaction was then immediately quenched through the addition of SDS–PAGE sample buffer containing 23 mM BME Samples were heated to 50 °C for 5 min before being loaded on 12% Novex WedgeWell Tris-Glycine gels (Thermo Fisher Scientific) Protein bands were stained with an InstantBlue Coomassie protein stain (Abcam) and band intensity was quantified using ImageJ (v The results were analysed in Excel and plotted using GraphPad Prism 9 For the experiments containing KorA in addition to KorB an equimolar amount was used (8 µM of WT or mutants The reaction was otherwise assembled identically and loaded on 4–20% Novex WedgeWell Tris-Glycine gels (Thermo Fisher Scientific) for sufficient separation of KorA in the samples Band intensity was quantified using ImageJ and the results were analysed in Excel and plotted using GraphPad Prism 9 All ITC experiments were recorded using a MicroCal PEAQ ITC instrument (Malvern Panalytical) For protein–nucleotide binding experiments all components were in 10 mM Tris–HCl and 150 mM NaCl the calorimetric cell was filled with 20 μM dimer concentration of KorB (WT or mutant) and a single injection of 0.4 μl of 500 μM small-molecule nucleotides or 200 μM protein partner was performed first Injections were carried out at 150 s intervals with a stirring speed of 750 r.p.m The raw titration data were integrated and fitted to a one-site binding model using the built-in software of the MicroCal PEAQ ITC instrument Controls of ligand into buffer and buffer into protein were performed with no signal observed induction of KorA WT/Y84A required no additional IPTG About 10 or 50 ml of culture was pelleted and resuspended in 500 μl resuspension buffer (0.1 M sodium phosphate buffer pH 7.4 Cells were disrupted using sonication at 10 µm for 10 s and subsequently pelleted The supernatant was transferred to a fresh microcentrifuge tube and assayed for catechol 2,3-oxygenase activity Samples were diluted 1:10 in reaction buffer (0.1 M sodium phosphate buffer pH 7.4 200 μM catechol) and incubated at room temperature for 1 min before the absorbance at 374 nm was determined using a BioMate 3 spectrophotometer (Thermo Fisher Scientific) 200 ng total protein lysate was resuspended in 1× SDS–PAGE sample buffer and heated to 95 °C for 10 min before loading Denatured samples were run on 12% Novex WedgeWell gels (Thermo Fisher Scientific) at 150 V for 55 min Resolved proteins were transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (BioRad) and incubated with a 1:5,000 dilution of α-KorB primary antibody (Cambridge Research Biochemicals) or with 1:300 dilution of α-KorA Membranes were washed and subsequently probed with a 1:10,000 dilution of mouse α-rabbit HRP-conjugated secondary antibody (Abcam) Blots were imaged after incubation with SuperSignal West PICO PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using an Amersham Imager 600 (GE HealthCare) Loading controls of denatured 200 ng total protein lysate were run on 12% Novex WedgeWell gels (Thermo Fisher Scientific) at 150 V for 55 min and stained with InstantBlue Coomassie protein stain (Abcam) C-terminally His-tagged versions of KorB (A6C) and KorA (WT/Y84A variant) with an extra cysteine residue at the C-terminus were coupled to maleimide-conjugated Alexa Fluor (AF) 488 and 647 A6C was selected because it resides in a surface-exposed intrinsically disordered region at the N-terminal region of KorB His-tagged KorB (A6C) and His-tagged KorA-extra C were purified as described for the WT proteins About 250 µl of 50 µM KorB (A6C) or KorA-extra C were incubated with 0.3 mM tris-carboxyethyl phosphine for 30 min at room temperature in a buffer containing 10 mM Tris–HCl and 300 mM NaCl 6 µl of 30 mM AF488 or AF647 (dissolved in DMSO) was added and the reaction was incubated with rotation at 4 °C overnight The conjugate solution was then loaded onto a Superdex increase 200 pg 10/300 gel filtration column (pre-equilibrated with 10 mM Tris–HCl pH 8.0) to separate labelled KorB/A from unincorporated fluorophore AF-labelled KorB/A was pooled and concentrated before storage as described for WT KorB and KorA the original pUC19 plasmid (4,886 bp) with one of each site was enlarged by introducing a piece of DNA obtained from a lab plasmid This resulted in a larger plasmid (7,699 bp) which after digestion with appropriate restriction enzymes produces the central part of a magnetic tweezers DNA construct with centred OB and OA sites To increase the number of OB sites, two long oligonucleotides (Supplementary Table 4) containing 2×OB sites separated by 40 bp with a PshAI restriction site in the middle of this region were annealed by heating at 95 °C for 5 min and cooling down to 20 °C at a rate of −1 °C min−1 in hybridization buffer (10 mM Tris–HCl pH 8.0 200 mM NaCl and 5 mM MgCl2) followed by a phosphorylation step of the 5′-terminal ends by the T4 PNK (NEB) This dsDNA duplex was ligated into the previous plasmid of 7,699 bp that already contained 1×OA and 1×OB sites digested with PshAI restriction enzyme (NEB) and dephosphorylated These oligonucleotides were designed to lose the original PshAI site at both ends after ligation so that once ligated into a cloning plasmid they could not be cleaved again by PshAI The single bona fide PshAI site located in the middle of the duplex allows for repetition of the ligation process to be repeated as many times as desired in the cloning plasmid to add new pairs of OB sites Plasmids containing 1×OA site and up to 8×OB have been obtained following this procedure Note that in one of the rounds of cloning and by chance half of a previous duplex was lost during the ligation process and therefore the final plasmid contains 8×OB instead of 9×OB as expected A plasmid with 1×OA site and 16×OB was produced by PCR amplifying an 8×OB cassette with Phusion High-Fidelity DNA Polymerase (Thermo Scientific) (see Supplementary Table 4 for primer sequences) The PCR fragment was then digested with SpeI and XhoI (both from NEB) and ligated into the plasmid already containing 1×OA site and 8×OB copies previously digested with XbaI (NEB) and XhoI and dephosphorylated This resulted in a plasmid with 1×OA site and 16×OB sites (8,705 bp) and potentially positive colonies were then selected by colony PCR Plasmids were purified from the cultures using a QIAprep Spin Miniprep Kit (QIAGEN) analysed by restriction enzyme digestion and finally verified by Sanger sequencing This plasmid was subsequently used to produce a magnetic tweezers dsDNA construct The PCR fragment was digested with SpeI and XbaI and ligated into the large plasmid resulting in a large plasmid with 1×OA site and 8×OB sites (22,394 bp) The large DNA plasmid containing 8×OB sites and 2×OA sites (22,733 bp) was produced by inserting a new copy of the OA site into the previously described plasmid digested with NruI (NEB) A new 339 bp dsDNA fragment with a copy of the OA site was obtained by digestion of the pUC19 plasmid containing a single copy of the OB and OA sites with SfoI and HpaI (NEB) purified and ligated into the NruI-linearized large plasmid described before The plasmids were cloned and analysed as described before for magnetic tweezers plasmids looking for a final plasmid with the new OA site in the same orientation as the previous one These plasmids were subsequently used to prepare various C-Trap dsDNA constructs A computer-controlled stage allowed rapid displacement of the optical traps within a five-channel fluid cell allowing the transfer of the tethered DNA between different channels separated by laminar flow Channel 1 contained 4.38 µm streptavidin-coated polystyrene beads (Spherotech) Channel 2 contained the DNA substrate labelled with multiple biotins at both ends Both DNA and beads were diluted in 20 mM HEPES pH 7.8 A single DNA tether was assembled by first capturing two beads in channel 1 and fishing for a DNA molecule in channel 2 The tether was then transferred to channel 3 filled with reaction buffer (10 mM Tris pH 8 5 mM MgCl2 and 1 mM DTT) to verify the correct length of the DNA by force–extension curves The DNA was then incubated for 1 min in channel 4 filled with KorB and/or KorA proteins in reaction buffer and supplemented with 2 mM CTP as indicated To reduce the fluorescence background in single KorB diffusion measurements imaging was occasionally performed in channel 3 after protein incubation in channel 4 (as indicated in figure legends) All the fluorescence intensities in kymograms were normalized and the scales in the intensity profiles were adjusted for better visualization Scale bars in figures represent fluorescence intensity on the kymographs The system is equipped with three laser lines for confocal microscopy (488 the 488 nm laser was used to excite AF488–KorB and the 635 nm laser to excite AF647–KorA with emission filters of 500–525 nm and 650–750 nm Protein-containing channels were passivated with BSA (0.1% w/v in PBS) for 30 min before the experiment Kymographs were generated by single line scans between the two beads using a pixel size of 100 nm and a pixel time of 0.1 ms resulting in a typical time per line of 22.4 ms The confocal laser intensity at the sample was 2.2 µW for the 488 laser and 1.92 µW for the 635 laser Experiments were performed in constant-force mode at 15 pN Magnetic tweezers experiments were performed as follows The DNA sample containing 16×OB sites was diluted in 10 mM Tris–HCl pH 8.5 and 1 mM EDTA and mixed with 1-µm-diameter magnetic beads (Dynabeads Magnetic beads were previously washed three times with PBS and resuspended in PBS/BSA at a 1:10 dilution The DNA to bead ratio was adjusted to obtain as many single-tethered beads as possible we introduced the DNA–bead sample in a double-PARAFILM (Sigma)-layer flow cell and allowed them to sink for 10 min to promote the binding of the digoxigenin (DIG)-labelled end of the DNA to the anti-DIG glass-coated surface a force of 5 pN was applied to remove non-attached molecules from the surface The chamber was washed with ~500 µl of PBS before experiments Torsionally constrained molecules and beads containing more than a single DNA molecule were identified from their distinct rotation–extension curves and discarded for further analysis Force–extension curves were generated by measuring the extension of the tethers at decreasing forces from 5.5 pN to 0.002 pN The curves were first measured on naked DNA molecules and then the experiment was repeated using different concentrations of B subtilis ParB and KorB ± KorA in a reaction buffer (10 mM Tris pH 8 1 mM DTT and 0.1 mg ml−1 BSA) supplemented with 2 mM CTP Data were analysed and plotted using OriginPro (v The PkorA sequence was shortened to its minimal elements as a 100 bp DNA scaffold and synthesized as separate PAGE-purified top and bottom strand oligos (IDT) (Supplementary Table 6) The two strands were resuspended separately to 1 mM solutions in 10 mM Tris–HCl pH 8.0 The strands were mixed in a 1:1 molar ratio for a 500 μM dsDNA (final concentration) and were heated to 95 °C before being cooled down to 25 °C in a 1 °C stepwise decrease using a Thermocycler PCR machine (Eppendorf) The resulting dsDNA was assayed by 2% w/v agarose gel electrophoresis for purity and was quantified using a Qubit (Invitrogen) dsDNA broad-range quantification kit The top and bottom strands were synthesized and annealed as described for WT PkorA 100-bp DNA scaffold used in nMS coli Eσ70 binding to the PkorA DNA scaffolds 50 nM of PkorA DNA scaffold was mixed with 50 nM E coli Eσ70 and incubated at 37 °C for 5 min coli His10–PPX–RNAP with a fivefold molar excess of σ70 at 37 °C for 15 min (excess σ70 was not purified away) The Eσ70–DNA sample was then loaded onto a 4.5% native Tris–borate–EDTA (90 mM TBE pH 8.3) polyacrylamide gel and run at a constant current of 15 mA for 1.5 h at 5–10 °C in a cold room The gel was stained for dsDNA using GelRed (Biotium) with measured values subtracted of the background and normalized to an E.coli Eσ70–DNA only control (no repression) for values to be averaged among replicates (n of at least 3 in all repression conditions) Repression as percentage values was calculated as (1-normalized intensity) × 100% graphed and statistically analysed in GraphPad Prism using unpaired Welch’s t-tests Plots of normalized band intensities against time resulted in a double exponential curve basal noise-level signal) dominating at time points with low fractions of competitor-resistant open promoter complexes Due to the rapid decline in WT PkorA open complexes a correction was applied by subtracting values that occur at t ≥ 30 min to remove the slow-decaying component and derive the true half-life from the fast decay as a single exponential decay curve This correction was not applied to PkorA:λPR discriminator open complex values as the time points measured did not decline to noise-level signal The plots were presented on a semi-log scale with the fraction of competitor-resistant open promoter complexes on a log10 scale and time on a linear scale with single exponential decay trend lines fitted (WT PkorA with R2 = 0.7637; PkorA:λPR discriminator with R2 = 0.9934) Analysis and plotting were performed using Microsoft Excel (v Eσ70 was formed by mixing purified His10–PPX–RNAP and a 2.5-fold molar excess of σ70 and incubated for 15 min at 37 °C Eσ70 was buffer exchanged into 20 mM Tris–HCl pH 8.0 500 mM NaCl and 5 mM DTT (to remove most glycerol from protein storage buffer) by centrifugal filtration and purified on a Superose 6 Increase 10/300 GL column (Cytiva) pre-equilibrated in 50 mM Tris–HCl pH 8.0 The eluted Eσ70 was concentrated to ~10–12 mg ml−1 (~21–26 μM) by centrifugal filtration Purified Eσ70 was supplemented with glycerol to a final concentration of 20% w/v Frozen samples were thawed and reconstituted in various combinations Sample concentrations for conditions without Eσ70 used 2 μM WT PkorA DNA and 5 μM KorA/B factors 5.5 μM WT PkorA DNA and 2.5-fold molar excess (12.5 μM) of KorA/B factors 0.5 mM CTP was used to ensure saturation of KorB subtract curve 10; smooth charge state distribution Gaussian; degree of Softmax distribution (beta parameter) The expected masses for the component proteins of the Eσ70 include α 158,008.1 Da (includes one Mg2+ and two Zn2+ ions); ω (lost N-terminal methionine) The expected masses for the Kor proteins and the DNA scaffold used are KorA monomer 12,825.6 Da; KorB (lost N-terminal methionine) The observed mass deviations (calculated as the percentage difference between the measured and expected masses relative to the expected mass) ranged from 0.001% to 0.3% with a typical mass deviation of 0.12% Cell cultures for deep sequencing were grown in the same conditions as for promoter–xylE reporter assays Cells from 5 ml of these cultures were resuspended in 300 µl of Puregene cell lysis solution (Qiagen) before incubation at 50 °C for 10 min and the samples were mixed by inverting 25 times before incubation at 37 °C for 60 min The samples were cooled to room temperature and 100 µl of Puregene protein precipitation solution (Qiagen) was added Samples were briefly vortexed and incubated on ice for 10 min and protein precipitate was removed via centrifugation at 17,000 g for 10 min The supernatant was transferred to a fresh microcentrifuge tube containing 600 µl of isopropanol and mixed by inverting 50 times Precipitated DNA was pelleted via centrifugation and the pellet was washed with 600 µl of 70% ethanol before final centrifugation at 17,000 g for 1 min and the DNA pellet was resuspended in 100 µl of distilled water and the average read coverage of the chromosome and plasmids was used to calculate the plasmid copy number per chromosome for each sample Data were subsequently analysed and plotted using GraphPad Prism 9 Experiments were performed in triplicates unless stated otherwise No statistical method was used to pre-determine the sample size Details of statistical tests and the P values are reported in the legends of relevant figures Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article No custom code was used or developed for the analysis of data presented in this paper Long-range enhancer–promoter contacts in gene expression control Long-distance cooperative and antagonistic RNA polymerase dynamics via DNA supercoiling DNA supercoiling-mediated collective behavior of co-transcribing RNA polymerases Long-range effects in a supercoiled DNA domain generated by transcription in vitro Effect of growth rate and incC mutation on symmetric plasmid distribution by the IncP-1 partitioning apparatus Replication and partitioning of the broad-host-range plasmid RK2 Complete nucleotide sequence of Birmingham IncP alpha plasmids Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2 Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans Self-organization of parS centromeres by the ParB CTP hydrolase ParB-type DNA segregation proteins are CTP-dependent molecular switches The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes A CTP-dependent gating mechanism enables ParB spreading on DNA CTP switches in ParABS-mediated bacterial chromosome segregation and beyond Bacterial chromosome segregation by the ParABS system Rules and exceptions: the role of chromosomal ParB in DNA segregation and other cellular processes ParB partition proteins: complex formation and spreading at bacterial and plasmid centromeres Flexibility in repression and cooperativity by KorB of broad host range IncP-1 plasmid RK2 The hierarchy of KorB binding at its 12 binding sites on the broad-host-range plasmid RK2 and modulation of this binding by IncC1 protein Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751 Dissection of the switch between genes for replication and transfer of promiscuous plasmid RK2: basis of the dominance of trfAp over trbAp and specificity for KorA in controlling the switch Repression at a distance by the global regulator KorB of promiscuous IncP plasmids A single aromatic residue in transcriptional repressor protein KorA is critical for cooperativity with its co-regulator KorB Purification of KorA protein from broad host range plasmid RK2: definition of a hierarchy of KorA operators Conserved C-terminal region of global repressor KorA of broad-host-range plasmid RK2 is required for co-operativity between KorA and a second RK2 global regulator Relief of ParB autoinhibition by parS DNA catalysis and recycling of ParB by CTP hydrolysis promote bacterial centromere assembly CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS ParB proteins can bypass DNA-bound roadblocks via dimer-dimer recruitment CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer korA function of promiscuous plasmid RK2: an autorepressor that inhibits expression of host-lethal gene kilA and replication gene trfA Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes The trfB region of broad host range plasmid RK2: the nucleotide sequence reveals incC and key regulatory gene trfB/korA/korD as overlapping genes Compilation and analysis of Escherichia coli promoter DNA sequences Anatomy of Escherichia coli sigma70 promoters Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced strand separation of double helical DNA Initial events in bacterial transcription initiation rRNA promoter regulation by nonoptimal binding of sigma region 1.2: an additional recognition element for RNA polymerase Mechanism of transcription initiation and promoter escape by E Structural origins of Escherichia coli RNA polymerase open promoter complex stability Connecting the dots: key insights on ParB for chromosome segregation from single-molecule studies Whole-genome analysis of the chromosome partitioning and sporulation protein Spo0J (ParB) reveals spreading and origin-distal sites on the Bacillus subtilis chromosome Effects of the P1 plasmid centromere on expression of P1 partition genes The P1 plasmid-partition system synthesizes two essential proteins from an autoregulated operon is required for full repression of the sopAB operon Pseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif The ParB-parS chromosome segregation system modulates competence development in Streptococcus pneumoniae Chromosome segregation proteins of Vibrio cholerae as transcription regulators a key transcriptional regulator of Shigella virulence requires a CTP ligand for its regulatory activities The virulence regulator VirB from Shigella flexneri uses a CTP-dependent switch mechanism to activate gene expression a transcriptional activator of virulence in Shigella flexneri Transcription activation by a sliding clamp On the choreography of genome folding: a grand pas de deux of cohesin and CTCF Cohesin and CTCF control the dynamics of chromosome folding Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the β-subunit flap domain of Escherichia coli RNA polymerase coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation 6S RNA mimics B-form DNA to regulate Escherichia coli RNA polymerase xia2: an expert system for macromolecular crystallography data reduction How good are my data and what is the resolution CCP4i2: the new graphical user interface to the CCP4 program suite Highly accurate protein structure prediction with AlphaFold ColabFold: making protein folding accessible to all Coot: model-building tools for molecular graphics REFMAC5 for the refinement of macromolecular crystal structures Crystal structure of KorA bound to operator DNA: insight into repressor cooperation in RP4 gene regulation Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene Long DNA constructs to study helicases and nucleic acid translocases using optical tweezers Visualizing helicases unwinding DNA at the single molecule level fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions DNA stretching induces Cas9 off-target activity Modular magnetic tweezers for single-molecule characterizations of helicases Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules Twisting and stretching single DNA molecules Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD NIH image to ImageJ: 25 years of image analysis Native mass spectrometry analysis of affinity-captured endogenous yeast RNA exosome complexes Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles MetaUniDec: high-throughput deconvolution of native mass spectra and collaborative data analyses: 2024 update McLean, T. C. et al. KorB switching from DNA-sliding clamp to repressor mediates long-range gene silencing in a multi-drug resistance plasmid. Mendeley Data https://doi.org/10.17632/8cw3ygfssy.1 (2024) Download references Gruber and members of our laboratories for helpful comments and M Dillingham (University of Bristol) for providing purified B This work is supported by the Royal Society University Fellowship Renewal URF\R\201020 and the Lister Institute fellowship (T.B.K.L.); by the Wellcome Trust Investigator grant 221776/Z/2/Z (T.B.K.L and T.C.M.); by the Biotechnology and Biological Sciences Research Council-funded Institute Strategic Program Harnessing Biosynthesis for Sustainable Food and Health (HBio) (BB/X01097X/1); by grants from the National Institutes of Health R35 GM118130 (S.A.D.) P41 GM109824 and P41 GM103314 (B.T.C.); by grants PID2020-112998GB-I00 (F.M.-H.) funded by MICIU/AEI/10.13039/501100011033 PID2023-146255NB-I00 (F.M.-H.) funded by MICIU/AEI/10.13039/501100011033 and European Regional Development Fund (FEDER and by grant FJC2020-044824-I (F.B.-P.) funded by MICIU/AEI/10.13039/501100011033 and European Union Next Generation Recovery Transformation and Resilience Plan (EU/PRTR) We also thank Diamond Light Source for access to beamlines I04-1 and I03 under proposal MX25108 (D.M.L.) These authors contributed equally: Thomas C Consejo Superior de Investigaciones Científicas Clara Aicart-Ramos & Fernando Moreno-Herrero Laboratory of Mass Spectrometry and Gaseous Ion Chemistry The authors declare no competing interests Nature Microbiology thanks Sankar Adhya, Gert Bange and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Analysis of the interaction of KorB with CDP and CTPɣS by ITC KorB binding to CDP was qualitatively much weaker than to CTP but the data precluded the estimation of a binding affinity through curve fitting An omit difference map for CTPγS was calculated after removing the ligands from the final structure and re-refining to convergence at 2.3 Å resolution Shown are orthogonal views of the two ligands from the chain A-B dimer only together with their associated omit density displayed as a semi-transparent cyan surface contoured at 2.0 σ It was not possible to unambiguously assign the positions of the ligand sulfur atoms subtilis Noc were measured by continuous detection of released inorganic phosphates (see Methods) CTPase rates were measured at 1 mM concentrations of CTP and in the presence of 0.5 µM of cognate DNA duplexes Data are represented as mean values ± SD from five replicates Source data a, (left panel) KorB is cysteine-less and did not crosslink in the presence of BMOE. (right panel) The CTD of KorB is likely a constant dimerization domain, as judged by the crosslinking of KorB (K351C) variant. K351C is predicted to be crosslinkable based on symmetry-related interactions observed in the previously published crystal structure of KorB CTD (PDB: 1IGQ) SDS-PAGE analysis of BMOE crosslinking products of 8 µM of KorB (K351C) dimer (and variants) ± 0.5 µM 24 bp OB/scrambled OB DNA ± 1 mM CTP Quantification of the crosslinked fraction is shown below each representative image Data are represented as mean values ± SD from three replicates Sub-stoichiometric concentrations of OB are sufficient to promote crosslinking of KorB (S47C) SDS-PAGE analysis of BMOE crosslinking products of 8 µM of KorB (S47C) dimer + 1 mM CTP + increasing concentration of 24 bp OB DNA (from 1/128 to 2/1 OB-to-KorB molar ratio) A ratio of ~16-fold less OB DNA to KorB was sufficient to achieve maximal crosslinking and N146A on KorB and their impact on KorB’s ability to repress OB-proximal or distal promoters as judged by promoter-xylE reporter assays (top panel) Values shown are fold of repression a ratio of XylE activities from cells co-harboring a reporter plasmid and KorB-expressing plasmid to that of cells co-harboring a reporter plasmid and an empty plasmid (KorB-minus control) (bottom panel) Absolute values of XylE activities from the same assay An α-KorB immunoblot and loading controls (Coomassie-stained SDS-PAGE) from lysates of cells used in the same experiments are also shown below Source data AF488-KorB did not bind DNA lacking OB site (left panel) Representative cartoon showing a DNA containing an OA site (but no OB site) trapped between the two beads in the optical tweezers (upper right panel) A scan showing DNA trapped between two beads inside a protein channel containing 50 nM AF488-KorB and 2 mM CTP after a minute of incubation with AF488-KorB + CTP in the protein channel was subsequently transferred to a channel with buffer only a high background from the fluorescently labeled protein Determination of the diffusion constant of KorB (left panel) Representative KorB trajectories measured on the DNA (n = 111) (middle panel) Mean squared displacement (MSD) of KorB trajectories for different time intervals (∆t) (right panel) The diffusion constant of KorB (1.61 ± 0.12 µm2/s mean ± SEM) was calculated as half of the slope of the linear fit of MSD versus ∆t the 25th and 75th percentiles of the distribution and whiskers extending to data within 1.5× interquartile range Outliers are displayed as points beyond the whiskers Source data (left panel) Cartoon of the basic magnetic tweezers (MT) components and the layout of the experiment and a schematic representation of a DNA containing 1xOA and a 16xOB cluster The positions of OA and OB sites are represented to scale (right panel) Average force-extension curves of bare DNA molecules (n = 56) and in the presence of different dimer concentrations of KorB + 2 mM CTP (500 nM n = 13) or Bacillus subtilis BsParB + 2 mM CTP (500 nM and in the presence of 1 µM KorB + 1 µM KorA + 2 mM CTP (n = 10) Data are represented as mean values ± SEM from three replicates Source data SDS-PAGE analysis of BMOE crosslinking products of 8 µM of KorB (S47C) dimer ± 8 µM dimer concentration of KorA + 1 mM CTP ± 1 µM 24 bp DNA containing both OB and OA sites (OB_OA) or both scrambled OB site and OA (OBSCR_OA) or both OB and scrambled OA site (OB_OASCR) X indicates a crosslinked form of KorB (S47C) KorA can promote clamp-defective KorB (R117A) and KorB (N146A) variants to N-engage SDS-PAGE analysis of BMOE crosslinking products of 8 µM of KorB (S47C R117A) dimer and KorB (S47C N146A) dimer ± 8 µM of KorA dimer ± 0.5 µM 24 bp OB/scrambled (SCR) OB DNA ± 1 mM CTP X indicates a crosslinked form of KorB (S47C R117A) or KorB (S47C N146A) Substitutions Y84A on KorA or F249A on KorB eliminated KorA-KorB interaction Analysis of the interaction of KorB (WT or variant) with KorA (WT or variant) by ITC Source data The crystal structure of the KorA-KorB-DNA complex was determined at 2.7 Å resolution A series of omit difference maps were calculated by separately removing parts of the final structure and re-refining to convergence at 2.7 Å resolution the KorA dimer (magenta) and the DNA duplex (orange) are displayed as semi-transparent surfaces on a color-coded backbone trace of the structure contoured at 2.0 σ and shown as orthogonal views Close-up of a KorB-KorA interface with only the side chains of key residues displayed Also shown is omit difference density (semi-transparent cyan surface contoured at 2.0 σ) calculated for the model after the removal of these side chains and re-refining Further detail on the KorB-KorA interface with color-coded van der Waals dots illustrating intimate contact In addition to the hydrogen bonds highlighted in panel b the aromatic ring of Y84 in KorA makes pi-pi interactions with the E248-F249 peptide bond of KorB which is reciprocated by the aromatic ring of F249 in KorB making pi-pi interactions with the E80-H81 peptide bond of KorB These interactions are indicated by the pale yellow double-headed arrows KorA can block the diffusion of KorB on DNA (left panel) Schematic of the C-trap optical tweezers experiments where a DNA containing a cluster of 8xOB sites and 2xOA sites was tethered between two beads and scanned with a confocal microscope using 488 nm and 635 nm illumination (right panel) More representative kymographs showing the distribution of AF647-KorA and AF488-KorB in the presence of CTP along a DNA for the four cases described The frequency of occurrence for each case is indicated (the total number of recorded events n = 182) Kymographs were taken in a buffer-only channel to reduce fluorescence background following a 60 s incubation in the protein channel KorB increases the residence time of KorA at OA (left panel) Schematic of the C-trap optical tweezers experiments where a DNA containing two clusters of 8xOB sites and 1xOA site were tethered between two beads and scanned with a confocal microscope using 635 nm illumination (right panel) Representative kymograph showing the binding of AF647-labeled KorA either alone or in the presence of unlabeled KorB the binding of AF647-KorA in the presence of unlabeled KorB and 2 mM CTP the binding of AF647-KorA (Y84A) in the presence of unlabeled KorB and 2 mM CTP and the binding of AF647-KorA in the presence of unlabeled KorB (F249A) and 2 mM CTP Concentrations of proteins are shown above each representative image KorA residence time experiments were performed in the protein channel using unlabeled KorB and a lower concentration of AF-KorA to minimize fluorescence background Source data Immunoblots (α-KorB and α-KorA) and loading controls (Coomassie-stained SDS-PAGE) from lysates of cells used in the same experiments are also shown below Source data Representative urea-PAGE gel closeup on the abortive RNA product (5’-ApUpG*-3’; *radiolabelled on guanosine alpha-phosphate) transcribed in in vitro abortive initiation assays on WT PkorA and the PkorA:λPR discriminator mutant linear DNA scaffolds with different mixes of Eσ70 and/or fivefold excess KorA fivefold excess KorB and/or saturating CTP WT PkorA in vitro transcription repression of E coli RNAP:σ70 holoenzyme (Eco Eσ70) in the presence of fivefold excess KorA and/or KorB Experiments were repeated at least three times All values are normalized to holoenzyme only control as mean values ± SEM Eσ70 + KorB condition has n = 3 while the rest are n = 4 and this remains valid as repression quantities are normalized to a Eσ70-only control and background-corrected for each gel run and SEMs are considered in statistical analysis P value was calculated by an unpaired Welch’s t-test; * p ≤ 0.05 The P values are 0.1827 (KorA vs KorA + KorB) Deconvolved native mass spectra of Eσ70 (5 μM) with 2.5-fold excess KorA dimer electrospray ionized in buffer of 300 mM or 150 mM ammonium acetate pH 8.0 and 0.01% Tween-20 Source data Oligonucleotides used or generated in this study uncropped gel images and abortive initiation assay data Download citation DOI: https://doi.org/10.1038/s41564-024-01915-3 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science BehrendKorb family commits $565,000 to support engineering research at BehrendEndowment also will name the Korb Family Atrium in the Advanced Manufacturing and Innovation CenterA $565,000 gift from Penn State alumni William and Wendy Korb will create a permanent fund for research and innovative teaching in Penn State Behrend’s School of Engineering — A $565,000 gift from Penn State alumni William and Wendy Korb will create a permanent fund for research and innovative teaching in Penn State Behrend’s School of Engineering The funding will be used to develop new teaching approaches and to support research initiatives and professional development opportunities for faculty and students The gift also will fund improvements to the school’s research and teaching labs which expands the Korb family’s generous and consistent support of the School of Engineering will benefit students in two major ways,” Chancellor Ralph Ford said “It will provide immediate improvements to our facilities and equipment enhancing the undergraduate research experiences that help our graduates stand out It also will allow us to recruit and retain the highly engaged faculty members who shape those student experiences.” To recognize the continued support of the Korbs Penn State Behrend has named the lobby of the Advanced Manufacturing and Innovation Center the Korb Family Atrium The two-story space is a gateway to Behrend’s Knowledge Park an innovation hub that is home to 20 companies and nearly 500 employees The west wing of the AMIC building houses Behrend’s industrial engineering and mechanical engineering programs which are supported by a materials-science lab and cutting-edge instrumentation including an Icon XR atomic force microscope The east wing is occupied by industry partners a leader in LED lighting systems for commercial vehicles The new endowment extends a tradition of giving by the Korbs who have long supported the School of Engineering at Behrend and the College of Engineering at University Park William Korb attended Penn State Behrend for two years before earning a degree in industrial engineering at University Park in 1962 Wendy Korb attended Penn State and then earned her degree at Indiana University the couple established the Korb Family Trustee Scholarship in Engineering at Penn State Behrend they created three early-career professorships in industrial engineering — including a position at Behrend The three-year awards support engineering faculty at the start of their academic careers The William and Wendy Korb Early Career Professorship in Industrial Engineering at the Behrend College is currently held by Julia Zhao an assistant professor of industrial engineering She is the third faculty member to benefit from the Korb professorship the Korbs funded five additional scholarships “We have always felt that an investment in education is one of the best investments you can make,” said William Korb an Erie native who retired as president and CEO of Marconi Commerce Systems the world’s leading supplier of fuel dispensers credit card readers and point-of-sale devices for gasoline stations Alexander Korb is director of the Stanley Burton Center for Holocaust and Genocide Studies at the University of Leicester and a scholar of the Holocaust in southeastern Europe in History from Humboldt University in Berlin and received earlier research fellowships from Yad Vashem and the Imre Kertesz Kolleg at the Friedrich Schiller University Jena During his fellowship at the USC Shoah Foundation Center for Advanced Genocide Research Professor Korb explored the phenomenon of collaboration drawing from a number of country case studies in Eastern and Southeastern Europe He argued that we need to include Jewish perspectives in order to understand collaboration because Jews knew their collaborating neighbors much better than the Germans did The Visual History Archive gave him a better sense of the victims’ perspectives Professor Korb conducted research for his upcoming book A Multitude of Lethal Attacks: Collaboration and Mass Violence in Southeastern Europe The book will explore how the Holocaust intertwined with ethnic cleansing and civil war Copyright © 2025 University of Southern California In another case, Hegseth included his wife and personal lawyer in Signal conversations about this same operation. Additional controversy surrounded the resignations and firings of his staff members, who were reportedly fighting among themselves over prerogatives and access to the secretary and resources within the Pentagon bureaucracy are nothing new Since the creation of the Department of Defense in the aftermath of World War II and the early years of the Cold War competition among the military services for resources and influence has been a staple of Washington life The same goes for the friction between the top brass of the armed forces and the civilian leadership in the Department of Defense From the Truman administration to the Trump administration the complexity of defense decisionmaking delivers supreme challenges to the participants in the interagency policymaking process as they meet international threats with timely and effective responses Also important are the relationships between the defense secretary and the other key players in the National Security Council, including the secretary of state Hegseth has apparently been able to navigate this minefield successfully; there is not yet apparent friction or acrimony that would call for Presidential intervention or create inter-agency roadblocks over policymaking The Congress is another matter. If he wishes to be an efficient secretary, Hegseth clearly needs to mend some fences on Capitol Hill. Even some Republican senators opposed his confirmation and continued to utter public criticism about his performance in office especially after the controversy surrounding his Signal chats The media is another arena in which the Hegseth Pentagon needs to up its game. Hegseth’s press coverage has been unflattering, even allowing for the inevitable skepticism of the Washington press corps. A third group of Hegseth skeptics are retired military officers of senior rank whose frequent criticisms of his qualifications for office and decisionmaking understandably attract widespread attention.  Finally, Hegseth himself will have to look in a mirror and make fundamental decisions about his perception of his personal role. His past military service as a combat soldier certainly deserves respect. On the other hand, he is no longer a frontline fighter but an executive presiding over the world’s largest (and arguably, most important) corporation Senior leadership calls for superior knowledge of the strategic demands imposed by the challenges of the five warfighting domains: land senior management also requires the ability to demonstrate the “CAN” (cognitive Cognitive leadership entails the appropriate knowledge and expertise for the mission Affective leadership refers to a leader’s ability to inspire and motivate others Normative leadership means that the leader normally plays by the rules but dispenses with them only under extraordinary or emergency conditions With National Security Advisor Mike Waltz and his deputy now removed Hegseth has a new chance to overcome the challenges made apparent in his first few months in office he will sink into the metaphorical quicksand pit that comes with the office Stephen Cimbala is a Distinguished Professor of Political Science at Penn State Brandywine and the author of numerous books and articles on international security issues Lawrence Korb has held national security positions at several think tanks and served in the Pentagon in the Reagan administration Stay in the know with The National Interest newsletter © Copyright 2025 Center for the National Interest We use cookies to enhance your browsing experience We use cookies to help you navigate efficiently and perform certain functions You will find detailed information about all cookies under each consent category below The cookies that are categorised as "Necessary" are stored on your browser as they are essential for enabling the basic functionalities of the site We also use third-party cookies that help us analyse how you use this website and provide the content and advertisements that are relevant to you These cookies will only be stored in your browser with your prior consent You can choose to enable or disable some or all of these cookies but disabling some of them may affect your browsing experience Necessary cookies are required to enable the basic features of this site such as providing secure log-in or adjusting your consent preferences These cookies do not store any personally identifiable data Functional cookies help perform certain functionalities like sharing the content of the website on social media platforms Analytical cookies are used to understand how visitors interact with the website These cookies help provide information on metrics such as the number of visitors Performance cookies are used to understand and analyse the key performance indexes of the website which helps in delivering a better user experience for the visitors Advertisement cookies are used to provide visitors with customised advertisements based on the pages you visited previously and to analyse the effectiveness of the ad campaigns We have the address for the funeral home & the family on file If you're not happy with your card we'll send a replacement or refund your money Goldfinch Funeral Home is committed to the safety of the families we serve \u003ca href=\"/coronavirus-notice/\"\u003eRead More\u003c/a\u003e The family of Lynne Bunch Korb created this Life Tributes page to make it easy to share your memories Lynne Bunch Korb died peacefully at her home on Thu.. Made with love by funeralOne Thanks for visiting The use of software that blocks ads hinders our ability to serve you the content you came here to enjoy We ask that you consider turning off your ad blocker so we can deliver you the best experience possible while you are here Penn State alumni William and Wendy Korb donated $565,000 to the university The gift will create a permanent fund for research and innovative teaching in Penn State Behrend's School of Engineering.  The funding will be used to develop new teaching approaches and support research initiatives and professional development opportunities for faculty and students.  The gift will also fund improvements to the school's research and teaching labs.  which expands the Korb family’s generous and consistent support of the School of Engineering will benefit students in two major ways,” said Chancellor Ralph Ford “It will provide immediate improvements to our facilities and equipment It also will allow us to recruit and retain the highly engaged faculty members who shape those student experiences.” the university has named the lobby of the Advanced Manufacturing and Innovation Center the Korb Family Atrium.  The two-story space is a gateway to Behrend's Knowledge Park Penn State Behrend says the new endowment extends a tradition of giving by the Korbs who have long support the School of Engineering.  William Korb attend Penn State Behrend for two years before earning a degree in industrial engineering at University Park in 1962 Wendy attended Penn State and then earned her degree at Indiana University.  they created three early-career professorships in industrial engineering that included a position at Behrend.  The William and Wendy Korb Early Career Professorship in Industrial Engineering at Behrend College is currently held by Julia Zhao She is the third faculty member to benefit from the professorship.  More information on the funding can be found on Penn State Behrend's website. This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Music lovers unite with College of Lake County’s spring music concerts beginning in March Critically acclaimed Kristin Korb will be the guest artist at the 48th annual “Art of Jazz” concert series on Sunday Kristin Korb is a rare breed of musician who effortlessly blends her talents as a bassist and vocalist captivating listeners with her crystal-clear voice while expertly maneuvering the bass “We are excited to welcome bassist and vocalist Kristin Korb to CLC to perform with our award-winning jazz ensemble,” CLC music instructor Michael Flack said “Her time with the great bassist Ray Brown (with whom she made her recording debut) along with her impressive teaching resume at the University of Southern California combine to offer our voice and instrumental students as well as the Lake County community a unique opportunity to learn from an experienced performer and educator.” • 48th Annual “Art of Jazz” Concert Series: 4 p.m. To purchase tickets, visit jlcenter.clcillinois.edu or call (847) 543-2300 Tickets can also be purchased at the James Lumber Center for the Performing Arts Box Office 10 a.m All concerts take place on the Mainstage at the James Lumber Center for the Performing Arts with the exception of the free Jazz Combo spring concert Become a fan of the College of Lake County Music Department on Facebook @CollegeofLakeCountyMusicDepartment and Instagram @collegeoflakecountymusicdept By Stephen J. Cimbala, Lawrence J. Korb | August 9 Missile test silo construction at Vandenberg Space Force Base (Credit: Federation of American Scientists/Airbus via Google Earth) Enormous cost overruns in the Sentinel program have engendered a debate about how or if to go forward with a US intercontinental ballistic missile (ICBM) modernization program We see five potential paths forward that might reduce costs and maintain or even improve the United States’ strategic posture But to make the best military and financial choice the United States government will have to consider how an updated missile force relates to evolving technology in the space and cyber realms and the implications of decisions about ICBM modernization for nuclear arms control Questions have been raised about the cost overruns for the Sentinel ICBM modernization program which aims to replace the existing fleet of Minuteman III missiles beginning in the next decade Sentinel is one part of a plan to replace all three legs of the U.S nuclear strategic triad of land-based intercontinental ballistic missiles (ICBMs) submarine-launched ballistic missiles (SLBMs) deployed on fleet ballistic missile submarines (SSBNs) Columbia class SSBNs and upgraded Trident II D-5 missiles are intended for the next generation of sea-based strategic forces and the B-21 Raider advanced stealth bomber is already on track to replace both remaining B-52 and B-2 bombers in conventional and nuclear roles Alternatives for US ICBM modernization.[2] The first option for dealing with Sentinel’s cost overruns would involve canceling the entire Sentinel program and continuing to modernize and upgrade the existing Minuteman ICBM force The argument for this option contends that Minuteman upgrades would be much less costly than Sentinel and still adequate to fulfill ICBM mission requirements But the Air Force and other experts contended that Minuteman had already passed its dependable shelf life and a systemwide upgrade is unavoidable if the United States is to sustain a viable land-based strategic deterrent Continued dependence upon Minuteman would really leave the United States with a two and one-half modernized deterrent system instead of three dependable components A second option would be to move to a nuclear strategic dyad instead of a triad and depend on a deterrent of submarine-based weapons and strategic bombers One rationale for this argument involves the vulnerability of the silo-based ICBM force to a first strike An associated concern about the ICBM force’s potential vulnerability: US leaders might be tempted to premature alerts or prompt launch during a crisis Supporters of a US nuclear dyad also argue that survivable submarine and bomber forces could provide necessary coverage for prompt and delayed targets without need of the additional targeting ICBMs might provide proponents of ICBMs contend that a three-legged triad of nuclear launch systems provides synergistic survivability that cannot be accomplished by a dyad A prospective attacker against three systems must plan conservatively against three potential retaliatory forces deployed in entirely different basing modes A three-sided deterrent force also allows for more uncertainty with respect to future breakthroughs in technology future submersible drone swarms could make the undersea environment more transparent and nuclear missile submarines more vulnerable to discovery and attack than they have been future ICBMs would be deployed on mobile platforms instead of in silos Mobility would improve their chances of surviving a first strike and require a smaller number of deployed missiles to cover the same targeting requirements for example a force of 100 to 200 mobile ICBMs with two or three warheads assigned to each versus the current fleet of 400 Minuteman IIIs The United States could use its extensive road or rail network (or parts of both) for mobile ICBM deployments without having to get into the environmental and construction issues that doomed the Jimmy Carter administration’s MX/MPS system There would be challenges with respect to management of road or rail traffic and specialized mobile launch systems would be required But the basic principles of mobile road and rail mobility for ICBMs have been well understood for many years; both the former Soviet Union and Russia have deployed and now deploy mobile ICBMs Yet another option would be to deploy ICBMs in so-called deep underground basing ICBMs would no longer be the front end of a prompt retaliatory force they would be buried in mountainsides in the US West and rolled out for retaliation only after the first wave of an enemy attack had been completed They would be a type of doomsday force that is presumably untargetable and therefore guaranteed to respond much of the US prompt counterforce mission would now be assigned to the sea-based deterrent although the submarine missile force and bombers would also form part of the survivable retaliatory force The challenge for this option would be to provide for some enduring command and control in the aftermath of an attack that destroyed significant parts of the nuclear command A fifth option for the ICBM force would be “conventionalization” of strategic land-based missile launchers This would change the mission of that force into one of conventional global precision strike (CGPS) the ICBM force would be equipped with conventional weapons of various yields and improved accuracies across international distances Russia would doubtless claim that any missile of this type overflying its territory would be assumed to be nuclear armed but the United States could allow international inspections to verify the non-nuclear status of these launchers conventionally armed ICBMs would be more threatening to potential adversaries than nuclear-armed missiles A state that doubted American willingness to attack with a nuclear first strike against one or more of its military targets might conclude that an American president would more easily be persuaded to strike with precision conventional weapons avoiding the collateral damage attached to nuclear use Domain challenges to strategic stability: space and cyber. Options for a future ICBM force will have to be considered within the larger context of evolving technology related to deterrence. The domains of space and cyber now form part of the context for military planners.[3] A putative future nuclear attacker must first establish domain awareness and operational control over the requirements for space and cyber warfare Space assets provide the defender with early warning; command and communication; geolocation; and other capabilities necessary to functioning as a cohesive military organization Cyberwar provides a means of disabling and-or confusing the opponent’s decision-making process and paralyzing infrastructure necessary for timely military response to threats The first wave of any nuclear first strike will doubtless consist of threatening behavior in space and the activation of hostile malware previously implanted within enemy military and civilian computing networks Complete physical destruction of enemy space and cyber assets is not necessary; only a sufficient measure of confusion would be needed on the tail end of a crisis that is heading over the cliff into war Hypersonic weapons cast another shadow of concern over deterrence and crisis stability.[4] Hypersonic glide vehicles are already being deployed on some Russian ICBMs and other major powers can be expected to deploy similar weapons or hypersonic cruise missiles in their future arsenals hypersonics allow less time for political leaders and their military advisors to receive early warning and to decide upon an effective response before their nuclear forces or other targets are struck Awareness of this possible vulnerability may encourage hasty decision making or partial delegation of decisions to pre-programmed AI systems The concern about capabilities of hypersonics may also lead to a decision in favor of launch on warning instead of riding out the attack and retaliating The problem of hypersonics is also related to uncertainty in the space and cyber domains combined with expectations of hypersonic attacks against forces and command systems invites preemptive attack justified as a “defensive” move based on worst case assumptions given developments in Russian and Chinese nuclear strategy and Russian President Vladimir Putin’s recurrent threats of nuclear first use in the war against Ukraine the United States may be forced to expand its nuclear arsenal instead of simply modernizing it Absent a prompt peace settlement of the war in Ukraine New START may be permanently stalled or ditched in principle and possibly Chinese interests (officially recognized or otherwise) in avoiding nuclear war in forestalling expensive and unnecessary arms races and in containing global nuclear proliferation Between now and the official expiry date for New START the United States and Russia should conduct unofficial talks between their arms control and military experts avoid provocative demarches with respect to the possibility of nuclear first use in Europe and act together to discourage the emergence of new nuclear weapons states (e.g. A game plan for the future of strategic stability With regard to strategic nuclear arms control we outline this possible US post-New START Interim Holding Force for the time period 2026-2030: This notional force falls within current or prospective New START limits if the United States and Russia decide to reboot and extend that treaty beyond 2026 It should provide for assured retaliation against a Russian force that is similarly constrained or against a Chinese force that levels off at numbers of operationally deployed warheads on strategic launchers between 1,000 and 1,500 Any longer-term follow-on force to the interim force suggested above would have to take into account the possibility of a future combined Russian-Chinese attack but that possibility is not necessarily the most logical basis for realistic US planning China will be very cautious about getting into a nuclear war as Russia’s tail gunner: Chinese perceptions of their own national interests will determine Beijing’s nuclear deterrence and defense priorities the chaos that could result from an unconstrained digital misanthropy could create “analysis paralysis” followed by distorted perceptions of immediate danger and insufficient awareness of available alternatives The Sentinel ICBM system is one option among many for modernizing the ICBM force but it has inertia—based on support from members of Congress and from defense contractors—that will be difficult to reverse at least some of the ICBM force should be assigned to mobile basing to reduce incentives for prompt launch based on mistaken or ambiguous warning The war in Ukraine should not obviate US-Russian efforts to revive the New START and post-New START arms control discussions and a rising Chinese nuclear power must eventually be brought into strategic arms limitation talks—if not immediately about force reductions then at least about concepts of military strategy The space and cyber realms are no longer sidebars or afterthoughts for deterrence and arms control but candidates to be prime movers for rethinking nuclear stability in decades ahead [1] Additional expert commentary on Sentinel appears in: Hans M. Kristensen, Matt Korda, Eliana Johns, and Mackenzie Knight, “United States Nuclear Weapons, 2024,” Bulletin of the Atomic Scientists, May 7, 2024, https://thebulletin.org/premium/2024-05/united-states-nuclear-weapons-2024/ [2] For an overview of this issue, see: Lauren Caston, Robert S. Leonard, Christopher A. Mouton, Chad J.R. Ohlandt, S. Craig Moore, Raymond E. Conley, and Glenn Buchan, The Future of the U.S. Intercontinental Ballistic Missile Force (Santa Monica, California: RAND Corporation, 2014), https://www.rand.org/pubs/monographs/MG1210.html [4] This issue is discussed by US Air Force Col Stephen Reny in “Nuclear-Armed Hypersonic Weapons and Nuclear Deterrence,” Strategic Studies Quarterly [5] A useful assessment appears in Daryl G. Kimball, “Does the United States Need More Nuclear Weapons?” Arms Control Today, July – August 2024, https://www.armscontrol.org/act/2024-07/features/does-united-states-need-more-nuclear-weapons [6] For further discussion “The focus of US military efforts in outer space should be…arms control,” Bulletin of the Atomic Scientists The Bulletin elevates expert voices above the noise. But as an independent nonprofit organization, our operations depend on the support of readers like you. Help us continue to deliver quality journalism that holds leaders accountable. Your support of our work at any level is important we promise our coverage will be understandable Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" Δdocument.getElementById( "ak_js_2" ).setAttribute( "value" I am not quite sure how this mixed solution, w/o Sentinels, solves the various problems presented. I still prefer ditching silo-based ICBMs altogether. https://thebulletin.org/2021/10/bilateral-strategic-stability-what-the-united-states-should-discuss-with-russia-and-china/#post-heading US nuclear modernization is in trouble every portion is behind schedule and the nation is rapidly approaching a readiness crisis meaning they’re running out of time.Discussing alternative basing methods now is too late Decisions should have been made decades ago.Sentinel was always going to be a massive civil engineering project because the infra-strucutre pre-dates Minuteman III The legacy nuclear deterrenct has an expiration date and country is in deep trouble Stephen J. Cimbala is Distinguished Professor of Political Science at Penn State University, Brandywine and the author of numerous books and articles... Read More Lawrence J. Korb, a retired Navy captain, has held national security positions at several think tanks and served in the Pentagon in the Reagan... Read More Copyright © 2025 Bulletin of the Atomic Scientists. All rights reserved. Registered 501(c)(3). EIN: 36-2136497 Terms of UsePrivacy Policy Search"The 314", AHM's Downtown West Project Moves Closer to ConstructionChris StritzelMar 202 min read"The 314" a 29-story mass-timber apartment tower at 2033 Locust Rendering by Korb Architects.AHM’s transformative Downtown West project is moving closer to starting construction focused primarily on the 2100 block of Locust Street in Downtown West will see three old buildings redeveloped and three new buildings constructed The plans were originally unveiled in 2022 with the City approving a 10-year $145 Million tower will be among the tallest mass-timber buildings in the world 68% of the units will be 1 bedroom or 1 bedroom with a den Changes to the design include a new facade design for the parking podium and a overhauled facade for the west facing side of the tower The tower will be constructed on the lot located at 2033 Locust who specializes in mass-timber architecture is the architect for this component of the project "The 314" will be the tallest building in Downtown West and likely the tallest building between Downtown and the Central West End Construction is expected to begin on “The 314” either later this year or early next full design process commenced in January of this year Interior of a living room inside of "The 314" Rendering by Korb Architects.As per previous plans 2101 Locust will become creative office space 45 apartments will be built into the buildings at 2109-15 Locust new-construction apartment building will be built on the lot at 2125 Locust and will include 75 apartments and retail space a 113-unit apartment building with retail space will be constructed between 21st and 22nd Streets Between the Washington Avenue building and the historic buildings on Locust a 411-space parking garage will be constructed AHM’s intent is to set sail on the construction of the 2125 and Washington Avenue buildings some time this summer Arcturis is in charge of the design for 2125 Locust and 2100 Washington The development sits just one block north of City SC’s Energizer Park (formerly CityPark) The stadium removed what was the most visible remnant of the failed north-south distributor freeway and is now home to the CitySC campus along with other nearby investments from other developers aims to revitalize Downtown West and make it vibrant Infill and renovation projects go a long way in achieving this create a new anchor for future developments to grow off of Additional renderings of "The 314" and the new buildings at 2100 Washington and 2125 Locust are featured in the gallery below By Stephen J. Cimbala, Lawrence J. Korb | September 13 A screenshot taken from President Joe Biden's interview for CBS' 60 minutes broadcast on September 17 during which he warned Russian President Vladimir Putin against using tactical nuclear or chemical weapons after a series of military losses in Ukraine It would change the face of war unlike anything since World War II," Biden said As the Biden administration prepares to depart office an assessment of its policies with respect to nuclear issues and future challenges become increasingly important President Joe Biden will be succeeded by either Vice President Kamala Harris or former President Donald Trump Either future president will have to pick up the ball where Biden left it the ball isn’t rolling as smoothly as it should Three elements of Biden’s nuclear policies promise to particularly challenge his legacy: the usefulness of New START as a prospective framework for organizing US-Russian strategic nuclear arms control; the implications of China’s apparent determination to become a third nuclear superpower; and the potential game changers to a (so far) successful nuclear nonproliferation regime These perspectives could determine whether the nuclear legacy of Biden and his administration can last—or if events will outrun it in a matter of days or weeks The Russian invasion of Ukraine has created a hiatus in Russian-US negotiations on their bilateral New Strategic Arms Reduction Treaty (New START) and Russia has officially suspended its participation in the treaty (although Russia has stated its intention to remain within current New START limits on the numbers of treaty-permitted operationally deployed launchers and weapons) the growing military alliance between China and Russia combined with China’s apparent intention to become a third nuclear superpower holds the potential to accelerate the three-sided nuclear arms race and encourage the rise of additional nuclear weapons states it is not clear if current Russian and US strategic nuclear forces lend themselves to renewal and continuation of New START prior to its scheduled expiry date of February 2026 An extended or reborn New START will have to meet criteria for deterrence stability although it has not taken the additional step of abrogating the treaty The eclipse of New START is of course neither necessary nor desirable The United States and Russia have considerable Cold War and post-Cold War experience in nuclear arms control and other efforts to limit the nuclear arms race including disagreements and involvement of either party in proxy wars have frequently marked the atmosphere for discussion of nuclear issues between the two major nuclear powers: US-Soviet and then US-Russian nuclear discussions and arms control negotiations took place during the large-scale US military involvement in the Vietnam war; despite wars in the Middle East in which the Americans and the Soviets supported opposite sides; during the Soviet war in Afghanistan in the 1980s; and despite the hothouse atmosphere in the same decade in Europe surrounding competing NATO and Soviet deployments of intermediate range ballistic missiles and the challenges in managing the Cold War endgame sooner or later the currently ongoing war in Ukraine will have to be ended by a negotiated settlement that will involve concessions by both Ukraine and Russia Letting New START lapse without a successor regime would be also a tacit encouragement to existing nuclear weapons states to increase their reliance on deterrence self-help in Europe (and also in the Middle East and Asia) And the absence of ongoing nuclear arms control is a fast track into additional nuclear arms racing Some argue that when political relations between states are unfriendly then arms control is impossible and that when they are friendly arms control is unnecessary But this argument falls short of explanatory and prescriptive power Arms control is more necessary between potential rivals than it is among members of the same alliance or other security community No military planner in NATO is worried about the nuclear arsenals of Britain or France and few if any members of that alliance fear that other currently non-nuclear members of NATO will become a nuclear weapons state any time soon This is especially the case so long as the US nuclear guaranty of extended deterrence continues to apply to any and all NATO members threatened by nuclear coercion Putin’s repeated invocation of nuclear coercion to support Russia’s war in Ukraine is counterproductive to his aim of ending the war in Ukraine on the most favorable possible terms for Russia Repetitive threats of nuclear first use that are not followed by any actual changes in Russian military operational behavior (of the kind that are “telltales” for intelligence collectors in the United States and NATO) simply immunize opponents and other observers against additional fears of crossing the nuclear threshold Such behavior “normalizes” nuclear coercion in a way that is very dangerous creating doubts about any firebreaks that may exist between escalation of conventional war fighting and nuclear first use The result could be a NATO or Russian quantitative leap in long-range missile strikes or other seemingly tactical enhancements that are perceived by the other side as qualitative jumps into the destruction of strategically vital targets—and considered to deserve of a nuclear response NATO missile strikes against vital Russian targets in Crimea or Ukrainian air strikes using US-built F-16 aircraft against targets inside Russia could signal to the Russian leadership a willingness by NATO to escalate conventional war beyond previously understood boundaries As Nikolai Sokov has pointed out, while the Ukrainian invasion of Kursk resulted in a battle with Russian forces that probably will not lead to Russia using nuclear weapons against Ukraine, Russian escalation against NATO countries cannot be ruled out.[5] Along with the concern about Russia’s dubious mixture of conventional war fighting and nuclear coercion comes the additional concern about Ukraine’s expectation that NATO and Ukraine will inflict a strategic victory against Russia and expel Russia entirely from territories now occupied in eastern and southern Ukraine repeated claims that Ukraine could have won the war on the ground had NATO and the United States supplied sufficient numbers of long-range weapons in good time fly in the face of operational realities: Russia outnumbers Ukraine in available numbers of service personnel and in the industrial sinews for protracted conventional warfare including a robust and growing military industrial base Admittedly Russia’s military performance in the early stages of the war was disappointing in terms of its inflated initial war aims insufficient preparedness for modern combined arms combat But with the experience of the last two and a half years Russia has improved its command of the operational-tactical battlespace and its ability to integrate modern technology (including or C4ISR; precision strike; and air-ground coordination) with traditional principles of ground combat and fire discipline But Ukraine’s temporary success did not change the fundamentals of military combat under modern conditions: Other things being equal time favors Russia by wearing out the Ukrainian forces and the collective NATO will to resist—provided Putin does not stumble into an ill-advised nuclear first use that blows the lid off prior expectations Putin has already committed a political blunder by bringing about the enlargement of NATO into a membership of 32 countries with the accession of Finland and Sweden in 2023 and 2024 thereby increasing the borders between Russia and NATO member states that are possible locations for inadvertent US defense officials and most expert analysts have come to the conclusion that China’s plans to modernize its strategic nuclear deterrent and expand its size and diversity may seek to equal the forces of the United States and Russia A 2023 study group convened by the Center for Global Security Research at Lawrence Livermore National Laboratory noted that: For the first time in its nuclear history, the United States faces two major power adversaries armed with large and diverse nuclear forces, capable of challenging the United States and its allies in a limited regional war fought with conventional forces, and bound together by a hostility to US-led global and regional orders and the resolve to bring about their end.[9] Similarly, the Congressional Commission on the Strategic Posture of the United States warned that US strategy should no longer treat China’s nuclear forces as a “lesser included threat” and that the United States needs a nuclear posture capable of deterring both countries, simultaneously.[10] In addition with respect to China’s growing nuclear capabilities Even if experts seem to agree that China will attempt to join the United States and Russia in forming a trilateral club of nuclear superpowers by the mid-2030s or so does it mean that the United States should plan its future nuclear modernization on the assumption of arms racing against the sum of both Russian and Chinese nuclear expansion One argument for additional US force building would be that for credible deterrence against both Russia and China the United States must maintain capabilities for damage limitation against either or both Capabilities for damage limitation against larger numbers of Russian and Chinese nuclear missiles would theoretically require more US ICBMs or other early launching and fast flying components of the strategic nuclear triad Increased numbers of Russian and Chinese hypersonic weapons might create additional pressure for US compensation in damage limitation the United States might prefer to take another approach that is less potentially destabilizing: but defenses that could provide continental-wide protection against large scale attacks would require a qualitative leap forward in technology point defenses to protect components of the US strategic nuclear retaliatory force are within reach of currently available air and missile defense systems addressing the prospective challenges posed by a rising nuclear China together with Russia will require both offensive and defensive countermeasures for damage limitation and for deterrence stability But the United States will need to engage in nuclear arms control too—although perhaps at two levels through explicit agreements between the United States and Russia based on a whirligig of conference diplomacy and military-to-military expert exchanges One of the success stories for international relations since the Cold War has been the robustness of the nuclear nonproliferation regime Future challenges to that regime will test its resilience One such challenge is the current status of Iran as a threshold nuclear weapons state Iran has at least three overlapping political objectives Tehran sees itself as the potentially dominant power in the Middle East in competition with Saudi Arabia (and possibly Turkey) Iran wants to push back against the US military presence in the region primarily by waging war through its proxy terrorist organizations Iran wants to destroy Israel or weaken it to the maximum extent possible Iran does not necessarily desire a nuclear weapons capability for the purpose of launching a nuclear first strike on Israel would be deterrence against nuclear coercive diplomacy or first use by Israel in order to provide more room for conventional military adventurism on the part of Iran in other ways: either by demonstrative testing of nuclear capable launchers; demanding a droit de regard on regional issues in opposition to the views of Israel by tacit or explicit threat to make available nuclear materials and know-how to their proxies in Gaza Iran and the P-6 states (the five Permanent Members of the UN Security Council and Germany) signed the Iran nuclear deal (the Joint Comprehensive Plan of Action The agreement placed restrictions on Iran’s civilian nuclear program in return for sanctions relief The JCPOA went into effect in January 2016 but President Trump withdrew the United States from the agreement in 2018 the Biden administration has sought to renew the agreement during several years of negotiations; but the parties have remained far apart Hamas’ attack on Israel on October 7 and the resulting war in Gaza have exacerbated already tense relations between the United States and Iran as did Iran’s continuing support for Houthi rebels in Yemen and other proxy attacks on US forces deployed in Iraq and Syria Israel continues to define Iran’s nuclear weaponization as unacceptable from the standpoint of Israel’s national security and survival And Iran continues to position itself for the jump from “almost” to “actual” weaponization in a matter of months were Iran to actually join the nuclear club Saudi Arabia has already indicated that it would have no choice but to do likewise for which two issues loom large in the near future military planning and joint training exercises pose new challenges Russia and China of late have increased the frequency and intensity of their military exercises with respect to conventional warfare but how far this extends into the protocols for nuclear deterrence or nuclear use is more uncertain There is obvious disagreement between Russian and Chinese political leadership with respect to public discussion of nuclear employment policy Putin’s repeated references to the possibility of Russian nuclear first use in Ukraine or elsewhere in Europe have received no public support from China and President Xi has publicly expressed his opposition to any use of nuclear weapons in that conflict China has no interest in being drafted into a game of nuclear chicken between NATO and Russia Another potential obstacle to nuclear jointness in Russian and Chinese military planning is the latent possibility of future conflict between the two states over other issues including territorial disputes and hegemony in Central Asia There is also the issue of who is now the senior and who is the junior partner in their relationship Russia was assumed to be the senior partner in military capability and China the leader in economic size and potential As China’s nuclear arsenal grows to a size more approximate to that of Russia and-or the United States its larger military ambitions may collide with those of Russia despite their shared opposition to US global leadership Russia and China may also probably be wary of embracing North Korea’s extremist policies with respect to South Korea although they may sit back and enjoy using Kim Jong-un as a pit bull that keeps the United States and its Asian allies in a diplo-dither Shared nuclear planning or exercises with North Korea are not precluded for Russia or China but neither are they necessary for their security The third aspect of proliferation has to do with the possibility of new nuclear weapons states emerging from the ranks of those feeling threatened by regional rivals and-or concerned about the reach of America’s nuclear umbrella many in South Korea and in Japan look askance at China’s aspirations for nuclear superpower status combined with China’s already imposing economic strength and improving conventional forces South Korea and Japan must also be concerned about North Korea’s growing nuclear arsenal and ambitions These and other US allies might reflect on the experience of Ukraine which gave up its nuclear weapons after the demise of the Soviet Union and might now be second guessing that decision in view of later events For advocates of nuclear arms control and-or disarmament To obviate an Asian or Middle East leap into nuclear weapons status the United States must provide reassurance that its own nuclear forces are sufficient not only for its own protection but also for extended deterrence that protects allies That reassurance might require a qualitatively or quantitatively enhanced US strategic nuclear force posture that is at odds with arms control or disarmament objectives and Russia could cap their strategic nuclear modernizations at New START levels or thereabouts (that is 1,500 operationally deployed warheads on intercontinental or transoceanic launchers) with order-of-magnitude verification based on national technical means There would still be space and cyber to deal with but it does not seem impossible to set limits also on hardware that already provides for sufficiency and more in mass destruction Biden’s nuclear legacy will be one of continuation of preexisting plans for US strategic nuclear modernization But this trajectory has been increasingly called into question by political leaders and national security experts—and promises to remain as such in the years to come China’s nuclear modernization and the potential collaboration of China and Russia (and North Korea) in military and security policy do require superior US and allied intelligence collection and assessment nuclear strategizing demands more than force building US nuclear forces support the United States’ grand strategy and foreign policy in the largest sense but they are not a one-size-fits-all solution The question of “how much is enough?” has no unique answer: It depends on the nature of the threat and the priority of nuclear deterrence compared to other missions Capabilities for conventional deterrence and war fighting and for cyber dominance are equally important Contrary to the pessimistic assumptions of some arms control can play an important role here: It provides a means for understanding the strategic priorities of other powers for exerting influence over their decisions about nuclear modernization and for avoiding nuclear arms races or nuclear war based on misunderstandings and inadvertence must ensure that nuclear arms control is high on its foreign policy agenda [1] Treaty between the United States of America and the Russian Federation on Measures for the Further Reduction and Limitation of Strategic Offensive Arms (Washington, D.C.: U.S. Department of State, April 8, 2010), http://www.state.gov/documents/organization/140035.pdf [2] Xiaodon Liang, “Russia Links Nonstrategic Nuclear Exercises to Threats,” Arms Control Today, June 2024, https://www.armscontrol.org/act/2024-06/news/russia-links-nonstrategic-nuclear-exercises-threats [3] Hans M. Kristensen, Matt Korda, Eliana Johns and Mackenzie Knight, “United States Nuclear Weapons, 2024,” Bulletin of the Atomic Scientists, May 7, 2024, https://thebulletin.org/premium/2024-05/united-states-nuclear-weapons-2024/ [4] For an expert assessment, see: Laurel Baker, “Interview: Rose Gottemoeller on the precarious future of arms control,” Bulletin of the Atomic Scientists, July 29, 2024, https://thebulletin.org/2024/07/interview-rose-gottemoeller-on-the-precarious-future-of-arms-control/ [7] Crisis stability or first strike stability means that neither side would derive a significant advantage from striking first in the last resort (preemption) or premeditated attack Arms race stability means that one state’s deployed or proposed force components would not necessarily encourage another state’s offsetting countermeasures [8] See: Samuel Charap and Christian Curriden Options for Post – New START Arm Control with Russia (Santa Monica [9] Brad Roberts, et. al., China’s Emergence as a Second Nuclear Peer: Implications for U.S. Nuclear Deterrence Strategy (Livermore, Calif.: Lawrence Livermore National Laboratory, Spring 2023, p. 4, https://cgsr.llnl.gov/content/assets/docs/CGSR_Two_Peer_230314.pdf [10] Madelyn Creedon and Jon Kyl, Co-Chairs, America’s Strategic Posture: The Final Report of the Congressional Commission on the Strategic Posture of the United States (Washington, D.C.: October, 2023), Executive Summary, viii, https://www.usip.org/sites/default/files/America’s_Strategic_Posture_Auth_Ed.pdf [13] Ibid. See also: David E. Sanger, “Biden Approved Secret Nuclear Strategy Refocusing on Chinese Threat,” New York Times, August 20, 2024, https://www.nytimes.com/2024/08/20/us/politics/biden-nuclear-china-russia.html [14] Henry Sokolski, “Xi and Putin Are Building More Nukes: How to Compete,” in Sokolski, ed., China, Russia, and the Coming Cool War (Arlington, Va.: Nonproliferation Policy Education Center, June 2024), p. 13, http://npolicy.org/wp-content/uploads/2024/06/2402-China-Russia-Cool-War.pdf [15] Lawrence J. Korb and Stephen J. Cimbala, “The Future of Missile Defense,” The National Interest, February 9, 2024, https://nationalinterest.org/feature/future-missile-defense-209252 [16] Vipin Narang “Biden Approved Secret Nuclear Strategy Refocusing on Chinese Threat.” — Last year's hurricanes caused issues surrounding St Two sewage treatment facilities had to be turned off for days to protect them from storm surge The city is now investing $7 million to upgrade two sewage treatment facilities "It would be nice to be able to shower and not feel like I'm contaminating myself," said Barbara Korb "The first thing you notice is the sewer smell…" she said The city had to temporarily shut down both the Northeast and the Southwest water treatment plants during last year's hurricanes to protect the facilities during storm surge We had to go and use the showers at family's homes make sure we had enough bottled water for us and the dogs," said Korb The Northeast sewage treatment plant leaked after Hurricane Helene The city said the water is safe to use now but residents who live North of 30th Avenue North in St you feel like it's not safe to drink the water you're really afraid to drink it," said Korb "I would like to feel really good when I open my tap up and drink some water and make some coffee," said Sandra Lewis who also lives in St The Northeast and Southwest facilities will each receive $3.5 million for upgrades to prevent issues and protect them from storm surge during this hurricane season The Northeast facility was already undergoing a $70 million improvement project but the extra funding will be used to make more upgrades people can come down and feel like they can drink our water safely," said Korb and I feel like that's part of where the system failed" Henry Betsey Jr. is facing charges for marrying three Florida women in three different counties at the same time. Now saying the system that handles marriage licenses kept them in the dark Latest Pinellas County News from ABC Action News Report a typo the image of the cluttered home studio is common in hip-hop those genres are often affairs that are solitary to the point of solipsism he’s turned his hand to each of them and more This is because the music Korb makes is for video games in his work as the composer songwriter and audio director of indie godheads Supergiant games The image that started off this article was taken from the documentary Developing Hell A masterpiece in a career of masterpieces and my first exposure to their work and Super Mario are just as iconic as any of their characters or imagery until one moment when I was in my first playthrough of Hades Here’s a quick catch-up if you haven’t played the game son of Hades and rogue prince of the underworld Having got right royally sick of his dad’s lies and bullying In one of my many (many) travels through Tartarus I encountered a room much more homey than the rest of this lava-encrusted wasteland I hear a voice singing softly in the distance The beauty of both voice and song makes me do something I’ve never done in my decades as a gamer and it’s a song of spine-tingling beauty that made me seek out the soundtrack of a game for the first time in my life While instrumental music has never sat well with me although the speed metal guitar battle ‘The Unseen Ones’ is absolutely ludicrous fun what set Korb’s work on Hades apart was the songwriting There are a handful of songs sung in the game and all of them are phenomenal pieces in their own right and the climactic credits song ‘In The Blood’ all help to cement Hades‘ place as pretty much my all-time favourite video game A game so good I went back and played through the entire Supergiant back catalogue. I found that in every one of those games from Pyre to Transistor and especially debut effort Bastion Korb had put together an absolute knock-out song or three Most often with frequent collaborator and possessor of that incredible voice that hooked me back in Tartarus Korb’s soundtracks do so much to deepen each game’s atmosphere and immersion but the songs are where the characters come to life has been number one on my Spotify Wrapped two years running A mix of trip-hop and dustbowl blues that Alabama 3 would kill for …Coming Home plays over the credits of the game and speaks directly from the characters’ points of view To elaborate would be to spoil a game that is required material if you have any interest in games at all all I’ll say is that the moment I heard it I basically knew it was one of my favourite songs of all time Is a love of gaming necessary to appreciate Korb’s work I can’t say; I’m a little bit too deep to say with any certainty what I can say is that as a way of understanding how music can inform storytelling With the full release of Hades II fast approaching and yet more brilliant songs powering it there’s never been a better time to discover the heart and soul of one of the great game studios of our time The earth is finally awakening from its winter sleep amid the returning green and delicate new life come be awakened by the enchanting world-acclaimed flautist Sunday at the Chateau is April 27th at 3:00pm “Ron Korb mesmerizes his audiences with dynamic performances of beautiful original music and interesting arrangements His work is accessible but also introduces new sounds and instruments to delighted audiences.” has travelled around the world collecting bamboo and various indigenous world flutes Through his lifetime of global exploration Ron has developed a distinctive musical persona He is a 2025 Grammy Nominee in Best Global Music Performance for “Kashira” by Masa Takumi and Best New Age Album for “Break of Dawn” by Ricky Kej Ron has performed for heads of state around the world and toured China seven times He has shared the stage with Olivia Newton John British rock bands Renaissance and The Yardbirds He has also collaborated with many film makers Windrush Estate Winery is located at 3100 Concession Road 3 Adjala Your $45 ticket includes the concert and a reception with award-winning Windrush VQA wines and meeting the artist. Guests under 19 or over 90 are free. Proceeds help empower underserved kids at youthLEADarts.com To purchase tickets, visit Upcoming Events at www.WindrushEstateWinery.com or call Marilyn at 905-729-0011 Receive our stories and events directly to your inbox Just Sayin’ Caledon brings you stories about Caledon people Connect with us on social media using #AllAboutCaledon Member of the Canadian Association of Journalists Patti Foley was a Bolton resident from 1991 until 2016 A former Regional Councillor and a long-time community volunteer she remains passionate about Caledon’s issues non-profits and businesses and continues to share those stories through Just Sayin’ Caledon Connect with us on social media using #AllAboutCaledon Copyright © 2025. Designed by Dash. Administrated by Foley Web Dev Giuseppe Venezia: I’ve Been Waiting For You (GleAM Records AM7031) | Calgaréal: Vanishing Point (independent release) | Avishai Cohen: Brightlight (Naïve/Believe BLV8583) | Kristin Korb: Sweet Dreams (Giant Sheep Music GSM0932) | Scott Colley this month has been a treat for someone who appreciates the sound of the bass I have the new releases from Avishai Cohen Kristin Korb and Giuseppe Venezia in my pile for November and I can’t wait to jump into the deep lows that the instrument offers Lamborghini and Gucci have two things in common; all of them are Italian and all of them are as cool as a cucumber fresh from the fridge You can add another name to the list: Giuseppe Venezia well he might not be cool (I couldn’t possibly judge because I don’t know him personally) but the brand of jazz he oversees on the seven-track album that I currently have playing certainly is There are hints of Dave Brubeck on title track I’ve Been Waiting For You John Coltrane-style sax on Blue Bird and a more contemporary feel on closing track The Shortest Story Did I mention a bass solo track that opens the album but I will – it’s a wonderful introduction to the backbone of the album When a bassist is the chief of the band there is a worry that the bass might sit high in the mix propping up the rhythm with tasteful moments but when the spotlight turns towards it There is a lovely warmth to the whole production and the bass comes across like a trusted old friend This is a tight band; one you could stumble across in a basement jazz club in Europe where the suits are sharp and the cocktails are raised to toast the music It’s a really good collection of music which makes the most of the opportunity to branch out The music of Calgaréal is a combination of the styles of Calgary and Montréal I’m not familiar with either musical style so I press play with a little trepidation but on first impressions You have the drama and romance of Celtic music – the opening part of Cercle is straight from the postcard images of rural Ireland where the stout flows alongside stories and arguments while the whistles flutes and bodhran plays; but midway through the track we’re transported to another world a world where gypsy jazz rules the cobbles before a mixture of the two worlds meet and It welcomes anything into its arms and as long as it sounds good This album repeatedly throws everything on its head Just when you think you’ve got the puzzle solved it throws in a track like La Soupe with a straight drumbeat cool bass riff and vocals before we’re thrown into a violin solo section that could be playing beneath a scene from Les Miserables Il Lago Accento drifts into a straighter jazz groove (the double bass is the star of the show here and listen out for the John Coltrane style sax solo) proving again that you never feel sure in what direction the music will go I’m so pleased I was given this album to review: it’s such a deeply layered album yet it still knows its place and works very If the double bass was the star of the show on Vanishing Point it seems a good time to move onto one of modern jazz’s most celebrated double bass players Avishai Cohen is an influence on countless bassists and he balances great musicianship with the ability to write great music It’s everything that you expect from an artist of this standing here with pianist Guy Moskovich and drummer Roni Kaspi and also featuring saxophonist Yuval Drabkin punchy trebles (just listen to the bass openings on Hope or Liebestraum No3 for how easy the instrument seems) At times the album is energetic and powerful but this is countered with moments of calm such as tracks Hitragut and Dabkin the latter cruising along like a boat on a river There’s even time for a groovy vocal cover of the classic Summertime which sounds like it was recorded in 1960s San Francisco the pianist and drummer going full throttle the band is fantastic and is led so powerfully by a modern great This album can be summarised in very simple terms – it’s a collection of the greatest hits of Eurythmics in a jazz style That summary will attract those curious to hear how you can turn synth-heavy 80s pop music into jazz the Porter-style lyrics of There Must Be An Angel (“I walk into an empty room and suddenly my heart goes boom”) fit perfectly a lot of the success here is down to Korb herself weaving her way through the music beautifully – Here Comes The Rain Again is a smoky sultry and bluesy ode and as far from the original as you can get but it’s a highlight I should also mention that as well as being a fine singer (I imagine there have been comparisons to Diana Krall made in the past) Korb is an accomplished double bassist providing the muscle on the album Her walking line on Sisters Are Doin’ It For Themselves is superb swinging and singing in equal measure and giving a solid rhythmic feel to build upon and I think some of this comes down to the fact that it doesn’t feel like a covers album it feels like the songs were used as an ignition something to build from and create on rather than doing a chord-by-chord copy I can see myself reaching for this album again over the next few months and probably doing some digging into the discography of Kristin Korb Offering a change of pace from everything that has come before one that requires a dedicated 50 minutes or so of your time with nothing else going on find a good chair (and an even better sound system) and let go… bass and drums – at the very peak of their skills intuitive and clearly have been playing together long enough to second guess each other a track that opens with a poignant piano part before widening the picture and increasing the drama It’s a powerful start but one that acts as a hint of things to come Ellipsis and The Thicket build until we reach You Are a seven-minute track that displays everything that is good about this trio rising and falling from passionate strings into a bass solo then from piano solo into a reflective quiet before rising again for more energy from a saxophone solo and finally ending with vocals I would say that I’m a so-so fan of jazz trios I often feel that something is missing in the bare setup of a trio but what Colley Simon and Blade do here is use the trio setup as a foundation the vocals) but even when it is just the nucleus of the band it never feels bare I guess this is the benefit of knowing each other so well knowing when to add that extra beat or note This is a superb album played by superb musicians © Unless otherwise indicated, all content copyright Jazz Journal 1948-2025 The greatest challenge will be ensuring that Russia does not renew its appetite for swallowing the remainder of Ukraine at the first opportunity.