Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute.

The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.

Site occupancy and tunable luminescence of Eu2+ and Eu3+ coactivated KCaY (PO4)2:Eu phosphors.

Utilizing the structure characteristic of KCaY (PO4)2 crystal, the site distribution of Eu2+ in KCaY (PO4)2:Eu phosphor coactivated with Eu2+ and Eu3+ ions is tuned. Upon 393-nm excitation, the as-prepared phosphor exhibits a broadband emission of Eu2+ peaked at ~ 475 nm and a typical red emission of Eu3+ with a strong 5D0-7F1 emission at ~ 591 nm. The luminescence color of the phosphor can be adjusted from blue to green, white, yellow, and red. The increasing concentration of Sr2+ and Eu2+ results in a blue shifting of Eu2+ emission. The increasing concentration of Eu3+ results in a red shifting of Eu2+ emission and an enhanced red emission of Eu3+. The luminescence behaviors of the phosphors are analyzed in terms of the site distribution of Eu2+ and Eu3+. A single-phase white light emitting was achieved in KCaY (PO4)2:Eu phosphor upon UV and NUV light excitation, indicating that the phosphor has potential application in white lighting.

Heterologous Expression of Ketoreductase ChKRED20 Mutant in Pichia pastoris and Bioreductive Production of (R)-1, 3-Butanediol.

(R)-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The ChKRED20 is a robust NADH-dependent ketoreductase identified from Chryseobacterium sp. CA49. We obtained a ChKRED20 mutant (M12) through directed evolutionary screening of ChKRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to (R)-1, 3-BDO. So far, both ChKRED20 and its mutants have been expressed in intracellular in E. coli, the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in P. pastoris, and the target protein yield was 302 mg/L in shake-flask fermentation and approximately 3.5 g/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 °C and had high activity within a broad pH range of 6.0-8.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to (R)-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of (R)-1, 3-BDO with excellent stereoselectivity (ee > 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of (R)-1, 3-BDO, and the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of ChKRED20 or its mutants, which may have applications in industrial settings.

A programmable pAgo nuclease with RNA target-cleavage specificity from the mesophilic bacterium Verrucomicrobia.

Argonaute (Ago) proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements. Almost all characterized pAgos prefer to cleave DNA targets. Here, we describe a novel pAgo from Verrucomicrobia bacterium (VbAgo) that can specifically cleave RNA targets rather than DNA targets at 37°C and function as a multiple-turnover enzyme showing prominent catalytic capacity. VbAgo utilizes DNA guides (gDNAs) to cleave RNA targets at the canonical cleavage site. Meanwhile, the cleavage activity is remarkably strengthened at low concentrations of NaCl. In addition, VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets, and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage. Moreover, VbAgo can efficiently cleave highly structured RNA targets at 37°C. These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.

Evolution of Cytochrome P450 Enzymes and Their Redox Partners in Archaea.

Cytochrome P450 monooxygenases (CYPs/P450s) and their redox partners, ferredoxins, are ubiquitous in organisms. P450s have been studied in biology for over six decades owing to their distinct catalytic activities, including their role in drug metabolism. Ferredoxins are ancient proteins involved in oxidation-reduction reactions, such as transferring electrons to P450s. The evolution and diversification of P450s in various organisms have received little attention and no information is available for archaea. This study is aimed at addressing this research gap. Genome-wide analysis revealed 1204 P450s belonging to 34 P450 families and 112 P450 subfamilies, where some families and subfamilies are expanded in archaea. We also identified 353 ferredoxins belonging to the four types 2Fe-2S, 3Fe-4S, 7Fe-4S and 2[4Fe-4S] in 40 archaeal species. We found that bacteria and archaea shared the CYP109, CYP147 and CYP197 families, as well as several ferredoxin subtypes, and that these genes are co-present on archaeal plasmids and chromosomes, implying the plasmid-mediated lateral transfer of these genes from bacteria to archaea. The absence of ferredoxins and ferredoxin reductases in the P450 operons suggests that the lateral transfer of these genes is independent. We present different scenarios for the evolution and diversification of P450s and ferredoxins in archaea. Based on the phylogenetic analysis and high affinity to diverged P450s, we propose that archaeal P450s could have diverged from CYP109, CYP147 and CYP197. Based on this study's results, we propose that all archaeal P450s are bacterial in origin and that the original archaea had no P450s.

Neuroprotective Effects of Erinacine A on an Experimental Model of Traumatic Optic Neuropathy.

Erinacine A (EA), a natural neuroprotectant, is isolated from a Chinese herbal medicine, Hericium erinaceus. The aim of this study was to investigate the neuroprotective effects of EA in a rat model of traumatic optic neuropathy. The optic nerves (ONs) of adult male Wistar rats were crushed using a standardized method and divided into three experimental groups: phosphate-buffered saline (PBS control)-treated group, standard EA dose-treated group (2.64 mg/kg in 0.5 mL of PBS), and double EA dose-treated group (5.28 mg/kg in 0.5 mL of PBS). After ON crush, each group was fed orally every day for 14 days before being euthanized. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined using flash visual-evoked potentials (fVEP) analysis, retrograde Fluoro-Gold labelling, and TdT-dUTP nick end-labelling (TUNEL) assay, respectively. Macrophage infiltration of ON was detected by immunostaining (immunohistochemistry) for ED1. The protein levels of phosphor-receptor-interacting serine/threonine-protein kinase1 (pRIP1), caspase 8 (Cas8), cleaved caspase 3 (cCas3), tumour necrosis factor (TNF)-α, tumour necrosis factor receptor1 (TNFR1), interleukin (IL)-1ß, inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), and superoxide dismutase 1 (SOD1) were evaluated by Western blotting. When comparing the standard EA dose-treated group and the double EA dose-treated group with the PBS-treated group, fVEP analysis showed that the amplitudes of P1−N2 in the standard EA dose group and the double EA dose-treated group were 1.8 and 2.4-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The density of RGC in the standard EA dose-treated group and the double EA dose-treated group were 2.3 and 3.7-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The TUNEL assay showed that the standard EA dose-treated group and the double EA dose-treated group had significantly reduced numbers of apoptotic RGC by 10.0 and 15.6-fold, respectively, compared with the PBS-treated group (p < 0.05). The numbers of macrophages on ON were reduced by 1.8 and 2.2-fold in the standard EA dose-treated group and the double EA dose-treated group, respectively (p < 0.01). On the retinal samples, the levels of pRIP, Cas8, cCas3, TNF-α, TNFR1, IL-1ß, and iNOS were decreased, whereas those of Nrf2, HO-1, and SOD1 were increased in both EA-treated groups compared to those in the PBS-treated group (p < 0.05). EA treatment has neuroprotective effects on an experimental model of traumatic optic neuropathy by suppressing apoptosis, neuroinflammation, and oxidative stress to protect the RGCs from death as well as preserving the visual function.

NO2 Sensing Capability of Pt-Au-SnO2 Composite Nanoceramics at Room Temperature.

Composite ceramics of metal oxides and noble metals have received much attention for sensing reducing gases at room temperature. Presently, composite ceramics of SnO2 and noble metals have been prepared and investigated for sensing oxidizing NO2 at room temperature. While dramatic increases in resistance were observed for both 1 wt% Pt-SnO2 and 5 wt% Au-SnO2 composite nanoceramics after being exposed to NO2 at room temperature, the largest increase in resistance was observed for 1 wt% Pt-5 wt% -Au-SnO2 composite nanoceramics among the three composites. The response to 0.5 ppm NO2--20% O2-N2 was as high as 875 at room temperature, with a response time of 2566 s and a recovery time of 450 s in the air of 50% relative humidity (RH). Further investigation revealed that water molecules in the air are essential for recovering the resistance of Pt-Au-SnO2 composite nanoceramics. A room temperature NO2-sensing mechanism has been established, in which NO2 molecules are catalyzed by Pt-Au to be chemisorbed on SnO2 at room temperature, and desorbed from SnO2 by the attraction of water molecules in the air. These results suggest that composite ceramics of metal oxides and noble metals should be promising for room temperature sensing, not only reducing gases, but also oxidizing gases.

Mitigating Electron Leakage of Solid Electrolyte Interface for Stable Sodium-Ion Batteries.

The interfacial stability is highly responsible for the longevity and safety of sodium ion batteries (SIBs). However, the continuous solid-electrolyte interphase(SEI) growth would deteriorate its stability. Essentially, the SEI growth is associated with the electron leakage behavior, yet few efforts have tried to suppress the SEI growth, from the perspective of mitigating electron leakage. Herein, we built two kinds of SEI layers with distinct growth behaviors, via the additive strategy. The SEI physicochemical features (morphology and componential information) and SEI electronic properties (LUMO level, band gap, electron work function) were investigated elaborately. Experimental and calculational analyses showed that, the SEI layer with suppressed growth delivers both the low electron driving force and the high electron insulation ability. Thus, the electron leakage is mitigated, which restrains the continuous SEI growth, and favors the interface stability with enhanced electrochemical performance.

Insights into Anion-Solvent Interactions to Boost Stable Operation of Ether-Based Electrolytes in Pure-SiOx ||LiNi0.8 Mn0.1 Co0.1 O2 Full Cells.

Ether solvents with superior reductive stability promise excellent interphasial stability with high-capacity anodes while the limited oxidative resistance hinders their high-voltage operation. Extending the intrinsic electrochemical stability of ether-based electrolytes to construct stable-cycling high-energy-density lithium-ion batteries is challenging but rewarding. Herein, the anion-solvent interactions were concerned as the key point to optimize the anodic stability of the ether-based electrolytes and an optimized interphase was realized on both pure-SiOx anodes and LiNi0.8 Mn0.1 Co0.1 O2 cathodes. Specifically, the small-anion-size LiNO3 and tetrahydrofuran with high dipole moment to dielectric constant ratio realized strengthened anion-solvent interactions, which enhance the oxidative stability of the electrolyte. The designed ether-based electrolyte enabled a stable cycling performance over 500 cycles in pure-SiOx ||LiNi0.8 Mn0.1 Co0.1 O2 full cell, demonstrating its superior practical prospects. This work provides new insight into the design of new electrolytes for emerging high-energy density lithium-ion batteries through the regulation of interactions between species in electrolytes.

Interfacial Evolution of the Solid Electrolyte Interphase and Lithium Deposition in Graphdiyne-Based Lithium-Ion Batteries.

All-carbon graphdiyne (GDY)-based materials have attracted extensive attention owing to their extraordinary structures and outstanding performance in electrochemical energy storage. Straightforward insights into the interfacial evolution at GDY electrode/electrolyte interface could crucially enrich the fundamental comprehensions and inspire targeted regulations. Herein, in situ optical microscopy and atomic force microscopy monitoring of the GDY and N-doped GDY electrodes reveal the interplay between the solid electrolyte interphase (SEI) and Li deposition. The growth and continuous accumulation of the flocculent-like SEI is directly tracked at the surface of GDY electrode. Moreover, the nanoparticle-shaped SEI homogeneously propagates at the interface when N configurations are involved, providing a critical clue for the N-doping effects of stabilizing interfaces and homogenizing Li deposition. This work probes into the dynamic evolution and structure-reactivity correlation in detail, creating effective strategies for GDY-based materials optimization in lithium-ion batteries.

Lifestyles Shape the Cytochrome P450 Repertoire of the Bacterial Phylum Proteobacteria.

For the last six decades, cytochrome P450 monooxygenases (CYPs/P450s), heme thiolate proteins, have been under the spotlight due to their regio- and stereo-selective oxidation activities, which has led to the exploration of their applications in almost all known areas of biology. The availability of many genome sequences allows us to understand the evolution of P450s in different organisms, especially in the Bacteria domain. The phenomenon that "P450s play a key role in organisms' adaptation vis a vis lifestyle of organisms impacts P450 content in their genome" was proposed based on studies on a handful of individual bacterial groups. To have conclusive evidence, one must analyze P450s and their role in secondary metabolism in species with diverse lifestyles but that belong to the same category. We selected species of the phylum Proteobacteria classes, Alpha, Beta, Gamma, Delta, and Epsilon, to address this research gap due to their diverse lifestyle and ancient nature. The study identified that the lifestyle of alpha-, beta-, gamma-, delta-, and epsilon-proteobacterial species profoundly affected P450 profiles in their genomes. The study determined that irrespective of the species associated with different proteobacterial classes, pathogenic species or species adapted to a simple lifestyle lost or had few P450s in their genomes. On the contrary, species with saprophytic or complex lifestyles had many P450s and secondary metabolite biosynthetic gene clusters. The study findings prove that the phenomenon mentioned above is factual, and there is no link between the number and diversity of P450s and the age of the bacteria.

Revealing the High Salt Concentration Manipulated Evolution Mechanism on the Lithium Anode in Quasi-Solid-State Lithium-Sulfur Batteries.

Lithium-sulfur batteries are promising candidates of energy storage devices. Both adjusting salt/solvent ratio and applying quasi-solid-state electrolytes are regarded as effective strategies to improve the lithium (Li) anode performance. However, reaction mechanisms and interfacial properties in quasi-solid-state lithium-sulfur (QSSLS) batteries with high salt concentration are not clear. Here we utilize in-situ characterizations and molecular dynamics simulations to unravel aforesaid mysteries, and construct relationships of electrolyte structure, interfacial behaviour and performance. The generation mechanism, formation process, and mechanical/chemical/electrochemical properties of the anion-derived solid electrolyte interphase (SEI) are deeply explored. Li deposition uniformity and dissolution reversibility are further tuned by the sustainable SEI. These straightforward evidences and deepgoing studies would guide the electrolyte design and interfacial engineering of QSSLS batteries.

Bridging Interparticle Li+ Conduction in a Soft Ceramic Oxide Electrolyte.

Li+-conductive ceramic oxide electrolytes, such as garnet-structured Li7La3Zr2O12, have been considered as promising candidates for realizing the next-generation solid-state Li-metal batteries with high energy density. Practically, the ceramic pellets sintered at elevated temperatures are often provided with high stiffness yet low fracture toughness, making them too brittle for the manufacture of thin-film electrolytes and strain-involved operation of solid-state batteries. The ceramic powder, though provided with ductility, does not yield satisfactorily high Li+ conductivity due to poor ion conduction at the boundaries of ceramic particles. Here we show, with solid-state nuclear magnetic resonance, that a uniform conjugated polymer nanocoating formed on the surface of ceramic oxide particles builds pathways for Li+ conduction between adjacent particles in the unsintered ceramics. A tape-casted thin-film electrolyte (thickness: <10 µm), prepared from the polymer-coated ceramic particles, exhibits sufficient ionic conductivity, a high Li+ transference number, and a broad electrochemical window to enable stable cycling of symmetric Li/Li cells and all-solid-state rechargeable Li-metal cells.

Micromechanism in All-Solid-State Alloy-Metal Batteries: Regulating Homogeneous Lithium Precipitation and Flexible Solid Electrolyte Interphase Evolution.

Sulfide-based solid-state electrolytes (SSEs) matched with alloy anodes are considered as promising candidates for application in all-solid-state batteries (ASSBs) to overcome the bottlenecks of the lithium (Li) anode. However, an understanding of the dynamic electrochemical processes on alloy anode in SSE is still elusive. Herein, in situ atomic force microscopy gives insights into the block-formation and stack-accumulation behaviors of Li precipitation on an Li electrode, uncovering the morphological evolution of nanoscale Li deposition/dissolution in ASSBs. Furthermore, two-dimensional Li-indium (In) alloy lamellae and the homogeneous solid electrolyte interphase (SEI) shell on the In electrode reveal the precipitation mechanism microscopically regulated by the alloy anode. The flexible and wrinkle-structure SEI shell further enables the electrode protection and inner Li accommodation upon cycles, elucidating the functional influences of SEI shell on the cycling behaviors. Such on-site tracking of the morphological evolution and dynamic mechanism provide an in-depth understanding and thus benefit the optimizations of alloy-based ASSBs.

Diversification of Ferredoxins across Living Organisms.

Ferredoxins, iron-sulfur (Fe-S) cluster proteins, play a key role in oxidoreduction reactions. To date, evolutionary analysis of these proteins across the domains of life have been confined to observing the abundance of Fe-S cluster types (2Fe-2S, 3Fe-4S, 4Fe-4S, 7Fe-8S (3Fe-4s and 4Fe-4S) and 2[4Fe-4S]) and the diversity of ferredoxins within these cluster types was not studied. To address this research gap, here we propose a subtype classification and nomenclature for ferredoxins based on the characteristic spacing between the cysteine amino acids of the Fe-S binding motif as a subtype signature to assess the diversity of ferredoxins across the living organisms. To test this hypothesis, comparative analysis of ferredoxins between bacterial groups, Alphaproteobacteria and Firmicutes and ferredoxins collected from species of different domains of life that are reported in the literature has been carried out. Ferredoxins were found to be highly diverse within their types. Large numbers of alphaproteobacterial species ferredoxin subtypes were found in Firmicutes species and the same ferredoxin subtypes across the species of Bacteria, Archaea, and Eukarya, suggesting shared common ancestral origin of ferredoxins between Archaea and Bacteria and lateral gene transfer of ferredoxins from prokaryotes (Archaea/Bacteria) to eukaryotes. This study opened new vistas for further analysis of diversity of ferredoxins in living organisms.

Characterization of the asexual developmental genes brlA and wetA in Monascus ruber M7.

Monascus spp. are widely used in the production of monacolin K and food- grade pigments in East Asia. In Aspergillus species, the three transcription factors BrlA â†’ AbaA â†’ WetA sequentially function as the central activators of asexual development (conidiation), leading to the formation of conidiophores. Unlike their close relative Aspergillus spp., Monascus spp. produce basipetospora-type asexual spores (conidia), and their genomes contain homologs of brlA and wetA but not abaA. In the present study, to investigate their roles in Monascus conidiation, MrbrlA and MrwetA were functionally characterized by gene knockout and overexpression in Monascus ruber M7. The results revealed that the deletion and overexpression of MrbrlA and/or MrwetA caused no apparent changes in the morphology, size, number, structure, or germination of conidia. However, deletion and overexpression of MrwetA severely repressed sexual development and affected the production of secondary metabolites. Taken together, these results suggest that the well-established central regulatory model of conidiation in Aspergillus is not applicable in their Monascus relatives. The results of the present study could enrich our understanding of the asexual development regulatory networks in filamentous fungi.

Set1-catalyzed H3K4 trimethylation antagonizes the HIR/Asf1/Rtt106 repressor complex to promote histone gene expression and chronological life span.

Aging is the main risk factor for many prevalent diseases. However, the molecular mechanisms regulating aging at the cellular level are largely unknown. Using single cell yeast as a model organism, we found that reducing yeast histone proteins accelerates chronological aging and increasing histone supply extends chronological life span. We sought to identify pathways that regulate chronological life span by controlling intracellular histone levels. Thus, we screened the histone H3/H4 mutant library to uncover histone residues and posttranslational modifications that regulate histone gene expression. We discovered 15 substitution mutations with reduced histone proteins and 5 mutations with increased histone proteins. Among these mutations, we found Set1 complex-catalyzed H3K4me3 promotes histone gene transcription and maintains normal chronological life span. Unlike the canonical functions of H3K4me3 in gene expression, H3K4me3 facilitates histone gene transcription by acting as a boundary to restrict the spread of the repressive HIR/Asf1/Rtt106 complex from histone gene promoters. Collectively, our study identified a novel mechanism by which H3K4me3 antagonizes the HIR/Asf1/Rtt106 repressor complex to promote histone gene expression and extend chronological life span.

Ancient Bacterial Class Alphaproteobacteria Cytochrome P450 Monooxygenases Can Be Found in Other Bacterial Species.

Cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins, are well-known players in the generation of chemicals valuable to humans and as a drug target against pathogens. Understanding the evolution of P450s in a bacterial population is gaining momentum. In this study, we report comprehensive analysis of P450s in the ancient group of the bacterial class Alphaproteobacteria. Genome data mining and annotation of P450s in 599 alphaproteobacterial species belonging to 164 genera revealed the presence of P450s in only 241 species belonging to 82 genera that are grouped into 143 P450 families and 214 P450 subfamilies, including 77 new P450 families. Alphaproteobacterial species have the highest average number of P450s compared to Firmicutes species and cyanobacterial species. The lowest percentage of alphaproteobacterial species P450s (2.4%) was found to be part of secondary metabolite biosynthetic gene clusters (BGCs), compared other bacterial species, indicating that during evolution large numbers of P450s became part of BGCs in other bacterial species. Our study identified that some of the P450 families found in alphaproteobacterial species were passed to other bacterial species. This is the first study to report on the identification of CYP125 P450, cholesterol and cholest-4-en-3-one hydroxylase in alphaproteobacterial species (Phenylobacterium zucineum) and to predict cholesterol side-chain oxidation capability (based on homolog proteins) by P. zucineum.

In Silico Analysis of P450s and Their Role in Secondary Metabolism in the Bacterial Class Gammaproteobacteria.

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.

Caseinolytic Proteins (Clp) in the Genus Klebsiella: Special Focus on ClpK.

Caseinolytic proteins (Clp), which are present in both prokaryotes and eukaryotes, play a major role in cell protein quality control and survival of bacteria in harsh environmental conditions. Recently, a member of this protein family, ClpK was identified in a pathogenic strain of Klebsiella pneumoniae which was responsible for nosocomial infections. ClpK is linked to the thermal stress survival of this pathogen. The genome wide analysis of Clp proteins in Klebsiella spp. indicates that ClpK is present in only 34% of the investigated strains. This suggests that the uptake of the clpk gene is selective and may only be taken up by a pathogen that needs to survive harsh environmental conditions. In silico analyses and molecular dynamic simulations show that ClpK is mainly α-helical and is highly dynamic. ClpK was successfully expressed and purified to homogeneity using affinity and anion exchange chromatography. Biophysical characterization of ClpK showed that it is predominantly alpha-helical, and this is in agreement with in silico analysis of the protein structure. Furthermore, the purified protein is biologically active and hydrolyses ATP in a concentration- dependent manner.