Emergence of Transferable mcr-9 Gene-Carrying Colistin-Resistant Salmonella enterica Dessau ST14 Isolated from Retail Chicken Meat in Korea.

Colistin is an important antibiotic currently used to manage infections caused by multidrug-resistant pathogens in both humans and livestock animals. A new mobile colistin-resistance (mcr-9) gene was recently discovered; this discovery highlighted the need for rigorous monitoring of bacterial resistance against colistin. Salmonella is one of the major pathogens responsible for foodborne illnesses; however, there is minimal information regarding the presence of mcr genes in foodborne Salmonella strains. The aim of this study was to investigate the presence of mcr genes among 178 Salmonella strains isolated from chicken meat in Korea. Antimicrobial susceptibility was measured using the broth microdilution method. Bioinformatics characterization of colistin-resistant strains and genetic environment of the mcr-9 gene were analyzed using next-generation sequencing. Transferability of the mcr-9 carrying colistin-resistant Salmonella strain was tested using broth-mating conjugation. Thirteen of the 178 Salmonella isolates showed colistin resistance, but only one strain, Salmonella Dessau ST14 (KUFSE-SAL043) from a traditional chicken market in Korea, carried an mcr family gene, mcr-9. This strain also carried other acquired antimicrobial resistance genes such as blaTEM-1B, qnrS1, and aac(6')-Iaa. Only the IncX1 plasmid replicon type was detected in this strain. In the strain KUFSE-SAL043, the mcr-9 gene was located between two insertion sequences, IS903B and IS26, followed by the downstream regulatory genes qseB-like and qseC-like, which were located between IS1R and ΔIS1R. Conjugation tests revealed that the mcr-9 gene was successfully transferred to Escherichia coli J53 at a mean frequency of 2.03 × 10-7. This is the first report of a transferable mcr-9 gene in Salmonella isolated from chicken meat in Korea, highlighting the possibility of transfer of colistin resistance. Therefore, the wide use of colistin should be reconsidered, and a One Health perspective should be adopted to monitor the antimicrobial resistance of Enterobacteriaceae strains in humans, livestock, and the environment.

Complete genome sequencing of Shigella sp. PAMC 28760: Identification of CAZyme genes and analysis of their potential role in glycogen metabolism for cold survival adaptation.

Shigella sp. PAMC 28760 (isolated from Himantormia sp. lichen in Antarctica) is a gram-negative, non-sporulating bacterium that has cellulolytic and amylolytic characteristics as well as glycogen metabolic pathways. In this study, we isolated S. sp. PAMC 28760 from Antarctic lichen, and present the complete genome sequence with annotations describing its unique features. The genome sequence has 58.85% GC content, 4,278 coding DNA sequences, 85 tRNAs, and 22 rRNA operons. 16S rRNA gene sequence analyses revealed strain PAMC 28760 as a potentially new species of genus Shigella, showing various differences from pathogenic bacteria reported previously. dbCAN2 analyses revealed 91 genes related to carbohydrate-metabolizing enzymes. S. sp. PAMC 28760 likely degrades polysaccharide starch to obtain glucose for energy conservation. This study provides a foundation for understanding Shigella survival adaptation mechanisms under extremely cold Antarctic conditions.

Mussel adhesive Protein-conjugated Vitronectin (fp-151-VT) Induces Anti-inflammatory Activity on LPS-stimulated Macrophages and UVB-irradiated Keratinocytes.

BACKGROUND: Skin inflammation and dermal injuries are a major clinical problem because current therapies are limited to treating established scars, and there is a poor understanding of healing mechanisms. Mussel adhesive proteins (MAPs) have great potential in many tissue engineering and biomedical applications. It has been successfully demonstrated that the redesigned hybrid type MAP (fp-151) can be utilized as a promising adhesive biomaterial. The aim of this study was to develop a novel recombinant protein using fp-151 and vitronectin (VT) and to elucidate the anti-inflammatory effects of this recombinant protein on macrophages and keratinocytes. METHODS: Lipopolysaccharide (LPS) was used to stimulate macrophages and UVB was used to stimulate keratinocytes. Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot. Inflammatory cytokines and NO and ROS production were analyzed. RESULT: In macrophages stimulated by LPS, expression of the inflammatory factors iNOS, COX-2, and NO production increased, while the r-fp-151-VT-treated groups had suppressed expression of iNOS, COX-2, and NO production in a dose-dependent manner. In addition, keratinocytes stimulated by UVB and treated with r-fp-151-VT had reduced expression of iNOS and COX-2. Interestingly, in UVB-irradiated keratinocytes, inflammatory cytokines, such as interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a, were significantly reduced by r-fp-151-VT treatment. CONCLUSIONS: These results suggest that the anti-inflammatory activity of r-fp-151-VT was more effective in keratinocytes, suggesting that it can be used as a therapeutic agent to treat skin inflammation.

Crystal structure of hypothetical protein PA4202 from Pseudomonas aeruginosa PAO1 in complex with nitroethane as a nitroalkane substrate.

Nitroalkane oxidase (NAO) and nitronate monooxygenase (NMO) are two different types of nitroalkane oxidizing flavoenzymes identified in nature. A previous study suggested that the hypothetical protein PA4202 from Pseudomonas aeruginosa PAO1 is NMO and utilizes only anionic nitronates. However, the structural similarity between the PA4202 protein and Streptomyces ansochromogenes NAO has motivated investigation for what features of the two enzymes differentiate between the NAO and NMO activities. Herein, we report the crystal structure of PA4202 in a ternary complex with a neutral nitroethane (NE) and flavin mononucleotide (FMN) cofactor to elucidate the substrate recognition mechanism using a site-directed mutagenesis. The ternary complex structure indicates that the NE is bound with an orientation, which is poised for the proton transfer to H183 (which is the essential first catalytic step with nitroalkanes), and subsequent reactions with FMN. Moreover, a kinetic study reveals that the catalytic reactions of the wild type and H183 mutants PA4202s with nitroalkane substrates may yield the products of hydrogen peroxide and nitrite that are specified to NAO, although they show a low catalytic efficiency. Our results provide the first structure-based molecular insight into the substrate binding property of the hypothetical protein PA4202, including the interactions with neutral nitroalkanes as the substrate.

Crystal structure of oleate hydratase from Stenotrophomonas sp. KCTC 12332 reveals conformational plasticity surrounding the FAD binding site.

Unsaturated fatty acids are toxic to various bacteria, causing their death or growth inhibition. To prevent this toxicity, unsaturated fatty acids should be converted into saturated fatty acids via hydrogenation reaction, which is the complete reduction of double bonds on the carbon chain. In a recent report, we observed that Stenotrophomonas sp. KCTC 12332 exhibited a high biotransformation activity of oleic acid (OA) in 10-hydroxystearic acid and identified the gene encoding oleate hydratase (OhySt) by complete genomic analysis. In the present study, to further investigate the structural features of OhySt, the recombinant protein was expressed in Escherichia coli, and then purified and crystallized. Biochemical assay showed that OhySt produces 10-hydroxystearic acid in a flavin adenosine dinucleotide (FAD)-dependent manner, indicating that it requires FAD as a cofactor. The OhySt structure, which is determined in its apo state, allows for a structural comparison with the previously reported FAD bound structure of oleate hydratase. The comparison of structures indicates remarkable conformational change of the loop region surrounding the FAD molecule upon binding of FAD. This change forces one of the important catalytic residues into position for catalysis.

Structural characterization of the full-length response regulator spr1814 in complex with a phosphate analogue reveals a novel conformational plasticity of the linker region.

Spr1814 of Streptococcus pneumoniae is a response regulator (RR) that belongs to the NarL/FixJ subfamily and has a four-helix helix-turn-helix DNA-binding domain. Here, the X-ray crystal structure of the full-length spr1814 in complex with a phosphate analogue beryllium fluoride (BeF3(-)) was determined at 2.0 Å. This allows for a structural comparison with the previously reported full-length unphosphorylated spr1814. The phosphorylation of conserved aspartic acid residue of N-terminal receiver domain triggers a structural perturbation at the α4-ß5-α5 interface, leading to the domain reorganization of spr1814, and this is achieved by a rotational change in the C-terminal DNA-binding domain.

Structural insight into the substrate inhibition mechanism of NADP(+)-dependent succinic semialdehyde dehydrogenase from Streptococcus pyogenes.

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the oxidation of succinic semialdehyde (SSA) to succinic acid in the presence of NAD(P)(+), and play an important role in the cellular mechanisms including the detoxification of accumulated SSA or the survival in conditions of limited nutrients. Here, we report the inhibitory properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) in a binary (ES) complex with SSA as the substrate and a ternary (ESS) complex with the substrate SSA and the inhibitory SSA, at 2.4 Å resolution for both structures. Analysis of the kinetic inhibitory parameters revealed significant substrate inhibition in the presence of NADP(+) at concentrations of SSA higher than 0.02 mM, which exhibited complete uncompetitive substrate inhibition with the inhibition constant (Ki) value of 0.10 ± 0.02 mM. In ES-complex of SpSSADH, the SSA showed a tightly bound bent form nearby the catalytic residues, which may be caused by reduction of the cavity volume for substrate binding, compared with other SSADHs. Moreover, structural comparison of ESS-complex with a binary complex with NADP(+) of SpSSADH indicated that the substrate inhibition was induced by the binding of inhibitory SSA in the cofactor-binding site, instead of NADP(+). Our results provide first structure-based molecular insights into the substrate inhibition mechanism of SpSSADH as the Gram-positive bacterial SSADH.

Structural basis for antifreeze activity of ice-binding protein from arctic yeast.

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed ß-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the ß-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common ß-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.

The structure of a shellfish specific GST class glutathione S-transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture.

Glutathione-S-transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H-site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H-site of LeGST may be the result of adaptation to their environments as sedentary organisms.

Crystal structure of the response regulator spr1814 from Streptococcus pneumoniae reveals unique interdomain contacts among NarL family proteins.

Spr1814 belongs to the NarL/FixJ subfamily of signal transduction response regulators (RR), and has been predicted to regulate the neighboring ABC transporter, which translocates antibiotic molecules in Streptococcus pneumoniae. Here, we report the crystal structure of full-length unphosphorylated spr1814 at 1.7Å resolution. The asymmetric unit contains two spr1814 molecules, which display very different conformations. Through comparisons with other RRs structures, we concluded that one molecule adopts a general inactive conformation, whereas the other molecule adopts an intermediate conformation. The superposition of each molecule showed that rotational change of the effector domain occurred in intermediate conformational state, implying that domain rearrangement could occur upon phosphorylation.

Characterization of pneumolysin from Streptococcus pneumoniae, interacting with carbohydrate moiety and cholesterol as a component of cell membrane.

The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MßCD, which is in a 3ß configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1-3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins.

Expression, purification and preliminary X-ray crystallographic analysis of nitroalkane oxidase (NAO) from Pseudomonas aeruginosa.

Nitroalkane oxidase (NAO) is a flavin-dependent enzyme which catalyses the oxidation of nitroalkanes to the corresponding aldehydes or ketones, nitrite and hydrogen peroxide. In order to better understand the structure and function of this enzyme, NAO from Pseudomonas aeruginosa was purified and crystallized as a native and a selenomethionine-substituted (SeMet) enzyme. Both crystals diffracted to a resolution of 1.9 Å and belonged to the primitive orthorhombic space group P21, with unit-cell parameters a = 70.06, b = 55.43, c = 87.74 Å, ß = 96.56° for native NAO and a = 69.89, b = 54.83, c = 88.20 Å, ß = 95.79° for SeMet NAO. Assuming the presence of two molecules in the asymmetric unit in both crystals, the Matthews coefficients (VM) for native and SeMet NAO were calculated to be 2.30 and 2.48 ų Da⁻¹, with estimated solvent contents of 46.50 and 50.37%, respectively.

Crystal structure of receiver domain of putative NarL family response regulator spr1814 from Streptococcus pneumoniae in the absence and presence of the phosphoryl analog beryllofluoride.

Spr1814 of Streptococcus pneumoniae is a putative response regulator (RR) that has four-helix helix-turn-helix DNA-binding domain and belongs to the NarL family. The prototypical RR contains two domains, an N-terminal receiver domain linked to a variable effector domain. The receiver domain functions as a phosphorylation-activated switch and contains the typical doubly wound five-stranded α/ß fold. Here, we report the crystal structure of the receiver domain of spr1814 (spr1814(R)) determined in the absence and presence of beryllofluoride as a phosphoryl analog. Based on the overall structure, spr1814(R) was shown to contain the typical fold similar with other structures of the receiver domain; however, an additional linker region connecting the receiver and DNA-binding domain was inserted into the dimer interface of spr1814(R), resulting in the formation of unique dimer interface. Upon phosphorylation, the conformational change of the linker region was observed and this suggests that domain rearrangement between the receiver domain and effector domain could occur in full-length spr1814.

Crystallization and preliminary X-ray crystallographic studies of a new class of enoyl-(acyl-carrier protein) reductase, FabV, from Vibrio fischeri.

Enoyl-(acyl-carrier protein) reductase (ENR) catalyzes the last step of the fatty-acid elongation cycle of the bacterial fatty-acid biosynthesis (FAS II) pathway. Recently, a new class of ENR has been identified from Vibrio cholerae and was named FabV. In order to understand the molecular mechanism of the new class of ENR at the structural level, FabV from V. fischeri was overexpressed, purified and crystallized. Diffraction data were collected to 2.7 Å resolution from a native crystal. The crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 123.53, b = 164.14, c = 97.07 Å. The presence of four molecules of FabV in the asymmetric unit gave a V(M) value of 2.81 Å(3) Da(-1), with a corresponding solvent content of 54.5%.

Crystallization and preliminary X-ray crystallographic studies of succinic semialdehyde dehydrogenase from Streptococcus pyogenes.

Succinic semialdehyde dehydrogenase (SSADH) plays a critical role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and catalyzes the NAD(P)(+)-coupled oxidation of succinic semialdehyde (SSA) to succinic acid (SA). SSADH from Streptococcus pyogenes has been purified and crystallized as the apoenzyme and in a complex with NAD(+). The crystals of native and NAD(+)-complexed SSADH diffracted to resolutions of 1.6 and 1.7 Å, respectively, using a synchrotron-radiation source. Both crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 93.3, b = 100.3, c = 105.1 Å for the native crystal and a = 93.3, b = 100.3, c = 105.0 Å for the complex crystal. Preliminary molecular replacement confirmed the presence of one dimer in both crystals, corresponding to a Matthews coefficient (V(M)) of 2.37 Å(3) Da(-1) and a solvent content of 48.0%.

Structural and kinetic analysis of free methionine-R-sulfoxide reductase from Staphylococcus aureus: conformational changes during catalysis and implications for the catalytic and inhibitory mechanisms.

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys(102) functions as the catalytic residue and Cys(68) as the resolving Cys that forms a disulfide bond with Cys(102). Cys(78), previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97-106 containing the catalytic Cys(102). We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.

Crystal structure of PduO-Type ATP:Cob(I)alamin adenosyltransferase from Bacillus cereus in a complex with ATP.

ATP:Cobalamin adenosyltransferases catalyze the transfer a 5'-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co-C bond of coenzyme B(12). There are three types of adenosyltransferases, CobA, PduO and EutT. Among these adenosyltransferases, the PduO-type adenosyltransferases is the most widely distributed enzyme. Structural comparisons between apo BcPduO and BcPduO in complex with MgATP revealed that the N-terminal strands of both structures were ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Furthermore, unlike other reported structures, apo BcPduO was bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site. This study provides more structural information into the role of active site residues on substrate binding.

Crystallization and preliminary X-ray crystallographic studies of enoyl-acyl carrier protein reductase (FabI) from Psuedomonas aeruginosa.

During fatty-acid biosynthesis, enoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the reduction of trans-2-enoyl-ACP to fully saturated acyl-ACP via the ubiquitous fatty-acid synthase system. NADH-dependent enoyl-ACP reductase (FabI) from Pseudomonas aeruginosa has been purified and crystallized as an apoenzyme and in a complex form with NADH and triclosan. Triclosan is an inhibitor of FabI and forms a stable ternary complex in the presence of NADH. The crystals of native and complexed FabI diffracted to resolutions of 2.6 and 1.8 Å, respectively. The crystals both belonged to space group P2(1), with unit-cell parameters a = 117.32, b = 155.844, c = 129.448 Å, ß = 111.061° for the native enzyme and a = 64.784, b = 107.573, c = 73.517 Å, ß = 116.162° for the complex. Preliminary molecular replacement further confirmed the presence of four tetramers of native FabI and one tetramer of the complex in the asymmetric unit, corresponding to Matthews coefficients (V(M)) of 2.46 and 2.05 Å(3) Da(-1) and solvent contents of 50.1 and 40.1%, respectively.

Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Arctic [correction of Aantarctic] yeast Leucosporidium sp. AY30.

Freezing is dangerous to cellular organisms because it causes an increase in the concentration of ions and other solutes in the plasma, denatures biomolecules and ruptures cell membranes. Some cold-adapted organisms can survive at subzero temperatures by producing proteins that bind to and inhibit the growth of ice crystals. To better understand the structure and function of these proteins, the ice-binding protein from Leucosporidium sp. AY30 (LeIBP) was overexpressed, purified and crystallized. The native crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a=b=98.05, c=106.13 Å. Since LeIBP lacks any cysteine or methionine residues, two leucine residues (Leu69 and Leu155) were substituted by methionine residues in order to obtain selenomethionine-substituted LeIBP for use in multiple-wavelength anomalous diffraction (MAD) phasing. The selenomethionine-substituted mutant crystallized in the same space group as the native protein.

Characterization of oxazolidinone and phenicol resistance genes in non-clinical enterococcal isolates from Korea.

OBJECTIVES: To investigate the distribution and genetic characteristics of linezolid-resistant enterococci. METHODS: Enterococcus faecalis and Enterococcus faecium strains were isolated from pigs, equipment, grounds, and employees of 19 Korean swine farms in 2017. Antimicrobial susceptibility testing was then performed and linezolid resistance genes were detected via PCR. For genetic epidemiological characterization, multilocus sequence typing and whole-genome sequencing data were analysed. RESULTS: Twenty-eightE. faecalis and five E. faecium strains were isolated from 1026 samples obtained from the 19 farms. Ten sequence types were identified among the E. faecalis strains, of which ST256 (42.9%) and ST86 (25%) were the most abundant. The oxazolidinone and phenicol resistance genes poxtA, optrA, and fexA were detected in isolates of E. faecalis (100%, 85.7%, and 67.9%, respectively) and E. faecium (100%, 60%, and 80%, respectively). The minimum inhibitory concentrations of linezolid in these isolates ranged from 2 mg/L to 12 mg/L. The whole-genome sequencing data indicated that fexA was located upstream of poxtA. CONCLUSIONS: This is the first study to report the detection of poxtA in isolates that were both susceptible and resistant to linezolid in Korea. These results demonstrate the importance of antimicrobial resistance monitoring programmes, including regular antimicrobial susceptibility testing and resistance gene expression analysis, to facilitate the control of the spread of antibiotic resistance in non-clinical settings in Korea.