Ki Hyun Nam

Envelope stress is a trigger of CRISPR RNA‐mediated DNA silencing in Escherichia coli

A widespread feature in the genomes of most bacteria and archaea is an array of clustered, regularly interspaced short palindromic repeats (CRISPRs) that, together with a group of CRISPR‐associated (Cas) proteins, mediate immunity against invasive nucleic acids such as plasmids and viruses. Here, the CRISPR‐Cas system was activated in cells expressing a plasmid‐encoded protein that was targeted to the twin‐arginine translocation (Tat) pathway. Expression of this Tat substrate resulted in upregulation of the Cas enzymes and subsequent silencing of the encoding plasmid in a manner that required the BaeSR two‐component regulatory system, which is known to respond to extracytoplasmic stress. Furthermore, we confirm that the CasCDE enzymes form a stable ternary complex and appear to function as the catalytic core of the Cas system to process CRISPR RNA into its mature form. Taken together, our results indicate that the CRISPR‐Cas system targets DNA directly as part of a defence mechanism in bacteria that is overlapping with but not limited to phage infection.

Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

Background: Cas2 is universally conserved and essential for new CRISPR spacer acquisition. Results: Bha_Cas2 uses a single metal ion to cleave dsDNA and is likely activated by a pH-dependent conformational change. A method to classify Cas2 into ssRNase and dsDNase is proposed. Conclusion: B. halodurans and T. thermophilus Cas2 are metal-dependent endonucleases. Significance: dsDNase activity is consistent with the direct involvement of Cas2 in new spacer acquisition. The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5′-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg2+ or Mn2+), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1–α1 loop.

Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca2+-dependent Double-stranded DNA Binding Activity

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca2+ was located at strategic positions in the oligomerization interface. We further showed that removal of Ca2+ ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca2+ ions.

Maxima in the thermodynamic response and correlation functions of deeply supercooled water

Pointing to a second critical point One explanation for the divergence of many of the thermodynamic properties of water is that there is a critical point in deeply supercooled water at some positive pressure. For bulk water samples, these conditions are described as “no mans land,” because ice nucleates before such temperatures can be reached. Kim et al. used femtosecond x-ray laser pulses to probe micrometer-sized water droplets cooled to 227 K (see the Perspective by Gallo and Stanley). The temperature dependence of the isothermal compressibility and correlation length extracted from x-ray scattering functions showed maxima at 229 K for H2O and 233 K for D2O, rather than diverging to infinity. These results point to the existence of the Widom line, a locus of maximum correlation lengths emanating from a critical point in the supercooled regime. Science, this issue p. 1589; see also p. 1543 Maxima in the isothermal compressibility and correlation length point to the existence of a second critical point in water. Femtosecond x-ray laser pulses were used to probe micrometer-sized water droplets that were cooled down to 227 kelvin in vacuum. Isothermal compressibility and correlation length were extracted from x-ray scattering at the low–momentum transfer region. The temperature dependence of these thermodynamic response and correlation functions shows maxima at 229 kelvin for water and 233 kelvin for heavy water. In addition, we observed that the liquids undergo the fastest growth of tetrahedral structures at similar temperatures. These observations point to the existence of a Widom line, defined as the locus of maximum correlation length emanating from a critical point at positive pressures in the deeply supercooled regime. The difference in the maximum value of the isothermal compressibility between the two isotopes shows the importance of nuclear quantum effects.

Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose.

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus xgiganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.

Nucleic acid binding surface and dimer interface revealed by CRISPR-associated CasB protein structures

TfuCasB1 and TfuCasB1 bind by molecular sieving (View Interaction: 1, 2)

A Mutation of the RNA Polymerase β′ Subunit (rpoC) Confers Cephalosporin Resistance in Bacillus subtilis

ABSTRACT In bacteria, mutations affecting the major catalytic subunits of RNA polymerase (encoded by rpoB and rpoC) emerge in response to a variety of selective pressures. Here we isolated a Bacillus subtilis strain with high-level resistance to cefuroxime (CEF). Whole-genome resequencing revealed only one missense mutation affecting an invariant residue in close proximity to the C-terminal DNA-binding domain of RpoC (G1122D). Genetic reconstruction experiments demonstrate that this substitution is sufficient to confer CEF resistance. The G1122D mutation leads to elevated expression of stress-responsive regulons, including those of extracytoplasmic function (ECF) σ factors (σM, σW, and σX) and the general stress σ factor (σB). The increased CEF resistance of the rpoCG1122D strain is lost in the sigM rpoCG1122D double mutant, consistent with a major role for σM in CEF resistance. However, a sigM mutant is very sensitive to CEF, and this sensitivity is still reduced by the G1122D mutation, suggesting that other regulatory effects are also important. Indeed, the ability of the G1122D mutation to increase CEF resistance is further reduced in a triple mutant strain lacking three ECF σ factors (σM, σW, and σX), which are known from prior studies to control overlapping sets of genes. Collectively, our findings highlight the ability of mutations in RNA polymerase to confer antibiotic resistance by affecting the activity of alternative σ factors that control cell envelope stress-responsive regulons.

Crystal structures of Dronpa complexed with quenchable metal ions provide insight into metal biosensor development.

Many fluorescent proteins (FPs) show fluorescence quenching by specific metal ions, which can be applied towards metal biosensor development. In this study, we investigated the significant fluorescence quenching of Dronpa by Co2+ and Cu2+ ions. Crystal structures of Co2+‐, Ni2+‐ and Cu2+ ‐bound Dronpa revealed previously unseen, unique, metal‐binding sites for fluorescence quenching. These metal ions commonly interact with surface‐exposed histidine residues (His194‐His210 and His210‐His212), and interact indirectly with chromophores. Structural analysis of the Co2+‐ and Cu2+‐ binding sites of Dronpa provides insight into FP‐based metal biosensor engineering.

Crystal structure of Thermoplasma acidophilum XerA recombinase shows large C‐shape clamp conformation and cis‐cleavage mode for nucleophilic tyrosine

Site‐specific Xer recombination plays a pivotal role in reshuffling genetic information. Here, we report the 2.5 Å crystal structure of XerA from the archaean Thermoplasma acidophilum. Crystallographic data reveal a uniquely open conformational state, resulting in a C‐shaped clamp with an angle of ~ 48° and a distance of 57 Å between the core‐binding and the catalytic domains. The catalytic nucleophile, Tyr264, is positioned in cis‐cleavage mode by XerAs C‐term tail that interacts with the CAT domain of a neighboring monomer without DNA substrate. Structural comparisons of tyrosine recombinases elucidate the dynamics of Xer recombinase.

Response to Comment on “Maxima in the thermodynamic response and correlation functions of deeply supercooled water”

Caupin et al. have raised several issues regarding our recent paper on maxima in thermodynamic response and correlation functions in deeply supercooled water. We show that these issues can be addressed without affecting the conclusion of the paper.