scholarly journals Structural and Biochemical Characterization of Rm3, a Subclass B3 Metallo-β-Lactamase Identified from a Functional Metagenomic Study

2016 ◽  
Vol 60 (10) ◽  
pp. 5828-5840 ◽  
Author(s):  
Ramya Salimraj ◽  
Lihong Zhang ◽  
Philip Hinchliffe ◽  
Elizabeth M. H. Wellington ◽  
Jürgen Brem ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Distinct Group ◽  
Wide Distribution ◽  
Sequence Comparisons ◽  
Content Type ◽  
Deep Channel ◽  
N Terminus ◽  
Environmental Reservoir ◽  
Metagenomic Study

ABSTRACTβ-Lactamase production increasingly threatens the effectiveness of β-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known β-lactamases and mobilization from environmental reservoirs. The spread of metallo-β-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other β-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant β-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene,blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure.blaRm3encodes a subclass B3 MBL that, when expressed in a recombinantEscherichia colistrain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by highKmvalues. An Rm3 crystal structure reveals the MBL superfamily αβ/βα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for β-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of β-lactam resistance.

scholarly journals Molecular and biochemical characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria

2005 ◽  
Vol 389 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Taisei KIKUCHI ◽  
Hajime SHIBUYA ◽  
John T. JONES
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biochemical Characterization ◽  
Glycosyl Hydrolase ◽  
Functional Characterization ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
Pinewood Nematode ◽  
Sequence Comparisons

We report the cloning and functional characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria. This is the first gene of this type from any nematode species. We show that a similar cDNA is also present in another closely related species B. mucronatus, but that similar sequences are not present in any other nematode studied to date. The B. xylophilus gene is expressed solely in the oesophageal gland cells of the nematode and the protein is present in the nematode's secretions. The deduced amino acid sequence of the gene is very similar to glycosyl hydrolase family 16 proteins. The recombinant protein, expressed in Escherichia coli, preferentially hydrolysed the β-1,3-glucan laminarin, and had very low levels of activity on β-1,3-1,4-glucan, lichenan and barley β-glucan. Laminarin was degraded in an endoglucanase mode by the enzyme. The optimal temperature and pH for activity of the recombinant enzyme were 65 °C and pH 4.9. The protein is probably important in allowing the nematodes to feed on fungi. Sequence comparisons suggest that the gene encoding the endo-β-1,3-glucanase was acquired by horizontal gene transfer from bacteria. B. xylophilus therefore contains genes that have been acquired by this process from both bacteria and fungi. These findings support the idea that multiple independent horizontal gene transfer events have helped in shaping the evolution of several different life strategies in nematodes.

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals Enrichment and Characterization of an Autotrophic Ammonia-Oxidizing Archaeon of Mesophilic Crenarchaeal Group I.1a from an Agricultural Soil

2011 ◽  
Vol 77 (24) ◽  
pp. 8635-8647 ◽  
Author(s):  
Man-Young Jung ◽  
Soo-Je Park ◽  
Deullae Min ◽  
Jin-Seog Kim ◽  
W. Irene C. Rijpstra ◽  
...  
Keyword(s):  
16S Rrna ◽  
Agricultural Soil ◽  
Ammonia Oxidizing Bacteria ◽  
Sequence Comparisons ◽  
Soil Nitrification ◽  
Content Type ◽  
Group I ◽  
Single Sequence

ABSTRACTSoil nitrification is an important process for agricultural productivity and environmental pollution. Though one cultivated representative of ammonia-oxidizingArchaeafrom soil has been described, additional representatives warrant characterization. We describe an ammonia-oxidizing archaeon (strain MY1) in a highly enriched culture derived from agricultural soil. Fluorescencein situhybridization microscopy showed that, after 2 years of enrichment, the culture was composed of >90% archaeal cells. Clone libraries of both 16S rRNA and archaealamoAgenes featured a single sequence each. No bacterialamoAgenes could be detected by PCR. A [13C]bicarbonate assimilation assay showed stoichiometric incorporation of13C intoArchaea-specific glycerol dialkyl glycerol tetraethers. Strain MY1 falls phylogenetically within crenarchaeal group I.1a; sequence comparisons to “CandidatusNitrosopumilus maritimus” revealed 96.9% 16S rRNA and 89.2%amoAgene similarities. Completed growth assays showed strain MY1 to be chemoautotrophic, mesophilic (optimum at 25°C), neutrophilic (optimum at pH 6.5 to 7.0), and nonhalophilic (optimum at 0.2 to 0.4% salinity). Kinetic respirometry assays showed that strain MY1's affinities for ammonia and oxygen were much higher than those of ammonia-oxidizing bacteria (AOB). The yield of the greenhouse gas N2O in the strain MY1 culture was lower but comparable to that of soil AOB. We propose that this new soil ammonia-oxidizing archaeon be designated “CandidatusNitrosoarchaeum koreensis.”

scholarly journals Dissection of the Hydrogen Metabolism of the EnterobacteriumTrabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Ute Lindenstrauß ◽  
Constanze Pinske
Keyword(s):  
Fossil Fuels ◽  
Biochemical Characterization ◽  
Model Organism ◽  
Attractive Alternative ◽  
Hydrogen Metabolism ◽  
Content Type ◽  
E Coli ◽  
Formate Hydrogenlyase ◽  
Alternative Carbon Source

ABSTRACTTrabulsiella guamensisis a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterizedEscherichia coliformate hydrogenlyase (FHL-2Ec) complex. TheT. guamensisFHL-2 (FHL-2Tg) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2Tgcomplex catalyzes the disproportionation of formate to CO2and H2. FHL-2Tghas activity similar to that of theE. coliFHL-1Eccomplex in H2evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2Tgoperon in the heterologousE. colihost has now enabled us to unambiguously prove FHL-2Tgactivity, and it allowed us to characterize the FHL-2Tgcomplex biochemically. Although the formate dehydrogenase (FdhH) genefdhFis not contained in the operon, the FdhH is part of the complex, and FHL-2Tgactivity was dependent on the presence ofE. coliFdhH. Also, in contrast toE. coli,T. guamensiscan ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2Tgand the H2-forming FHL-2Tgunder these conditions.IMPORTANCEBiological H2production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2producers, and the prototype FHL-1Eccomplex inE. coliis well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2Eccomplex, allow a first biochemical characterization ofT. guamensis’s fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.

scholarly journals Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

2015 ◽  
Vol 198 (3) ◽  
pp. 544-552 ◽  
Author(s):  
Manuel Osorio-Valeriano ◽  
Javier de la Mora ◽  
Laura Camarena ◽  
Georges Dreyfus
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Basal Body ◽  
Biochemical Characterization ◽  
High Molecular Mass ◽  
Content Type ◽  
Biochemical Characteristics ◽  
Outer Membranes ◽  
Architectural Features ◽  
Rotary Motor

ABSTRACTThe flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacteriumRhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted byin silicomodeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N- and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium.IMPORTANCEDespite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system ofR. sphaeroidesis presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled.

scholarly journals Biochemical characterization of the catalytic domains of three different clostridial collagenases

2009 ◽  
Vol 390 (1) ◽  
Author(s):  
Ulrich Eckhard ◽  
Esther Schönauer ◽  
Paulina Ducka ◽  
Peter Briza ◽  
Dorota Nüss ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Substrate Affinity ◽  
Catalytic Domains ◽  
Zinc Binding Site ◽  
N Terminus ◽  
Activation Mechanisms ◽  
Order Of Magnitude ◽  
Therapy And Diagnosis ◽  
Prime Target

Abstract Clostridial collagenases are used for a broad spectrum of biotechnological applications and represent prime target candidates for both therapy and diagnosis of clostridial infections. In this study, we biochemically characterized the catalytic domains of three clostridial collagenases, collagenase G (ColG) and H (ColH) from Clostridium histolyticum, and collagenase T (ColT) from C. tetani. All protein samples showed activity against a synthetic peptidic substrate (furylacryloyl-Leu-Gly-Pro-Ala, FALGPA) with ColH showing the highest overall activity and highest substrate affinity. Whereas the K m values of all three enzymes were within the same order of magnitude, the turnover rate k cat of ColG decreased 50- to 150-fold when compared to ColT and ColH. It is noteworthy that the protein N-terminus significantly impacts their substrate affinity and substrate turnover as well as their inhibition profile with 1,10-phenanthroline. These findings were complemented with the discovery of a strictly conserved double-glycine motif, positioned 28 amino acids upstream of the HEXXH zinc binding site, which is critical for enzymatic activity. These observations have consequences with respect to the topology of the N-terminus relative to the active site as well as possible activation mechanisms.

scholarly journals Legionella dresdenensis sp. nov., isolated from river water

2010 ◽  
Vol 60 (11) ◽  
pp. 2557-2562 ◽  
Author(s):  
Paul Christian Lück ◽  
Enno Jacobs ◽  
Isolde Röske ◽  
Ute Schröter-Bobsin ◽  
Roger Dumke ◽  
...  
Keyword(s):  
Type Strain ◽  
Biochemical Characterization ◽  
Novel Species ◽  
Phenotypic Characterization ◽  
Fatty Acid Profiles ◽  
Sequence Comparisons ◽  
River Elbe ◽  
Macrophage Infectivity Potentiator ◽  
Macrophage Infectivity

Legionella-like isolates, strains W03-356T, W03-357 and W03-359, from three independent water samples from the river Elbe, Germany, were analysed by using a polyphasic approach. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut glass colony appearance that grew only on l-cysteine-supplemented buffered charcoal yeast extract agar. Phylogenetic analysis based on sequence comparisons of the 16S rRNA, macrophage infectivity potentiator (mip), gyrase subunit A (gyrA), ribosomal polymerase B (rpoB) and RNase P (rnpB) genes confirmed that the three isolates were distinct from recognized species of the genus Legionella. Phenotypic characterization of strain W03-356T based on fatty acid profiles confirmed that it was closely related to Legionella rubrilucens ATCC 35304T and Legionella pneumophila ATCC 33152T, but distinct from other species of the genus Legionella. Serotyping of the isolates showed that they were distinct from all recognized species of the genus Legionella. Strains W03-356T, W03-357 and W03-359 are thus considered to represent a novel species of the genus Legionella, for which the name Legionella dresdenensis sp. nov. is proposed. The type strain is W03-356T (=DSM 19488T=NCTC 13409T).

scholarly journals Genetic and Biochemical Characterization of FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae

2015 ◽  
Vol 59 (12) ◽  
pp. 7420-7425 ◽  
Author(s):  
Laurent Dortet ◽  
Laurent Poirel ◽  
Samia Abbas ◽  
Saoussen Oueslati ◽  
Patrice Nordmann
Keyword(s):  
Biochemical Characterization ◽  
Enterobacter Cloacae ◽  
Conjugative Plasmid ◽  
Lower Sensitivity ◽  
Gene Encoding ◽  
Content Type ◽  
Class A ◽  
Encoding Gene ◽  
Lactamase Gene

ABSTRACTAnEnterobacter cloacaeisolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC50s) of clavulanic acid and tazobactam were 10-fold higher than those found forKlebsiella pneumoniaecarbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. TheblaFRI-1gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5′ end of the β-lactamase gene was identified, leading to inducible expression of theblaFRI-1gene.

Sign in / Sign up

Export Citation Format

Share Document