scholarly journals Biochemical Characterization of the Acquired Metallo-β-Lactamase SPM-1 from Pseudomonas aeruginosa

2003 ◽  
Vol 47 (2) ◽  
pp. 582-587 ◽  
Author(s):  
Tanya A. Murphy ◽  
Alan M. Simm ◽  
Mark A. Toleman ◽  
Ronald N. Jones ◽  
Timothy R. Walsh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Homology Model ◽  
Structure Characteristic ◽  
Site Structure ◽  
Wide Range ◽  
Zinc Chelator ◽  
Active Site Structure

ABSTRACT SPM-1 is a new metallo-β-lactamase recently identified in Pseudomonas aeruginosa strain 48-1997A, isolated in Sao Paulo, Brazil. Kinetic analysis demonstrated that SPM-1 has a broad hydrolytic profile across a wide range of β-lactam antibiotics. Considerable variation was observed within the penicillin, cephalosporin, and carbapenem subfamilies; however, on the whole, SPM-1 appears to preferentially hydrolyze cephalosporins. The highest k cat/ Km ratios (in micromolar per second) overall were observed for this subgroup. The hydrolytic profile of SPM-1 bears the most similarity to that of the metallo-β-lactamase IMP-1, yet for the most part, SPM-1 has k cat/Km values higher than those of IMP-1. Zinc chelator studies established that progressive inhibition of SPM-1 by EDTA, dipicolinic acid, and 1-10-o-phenanthroline demonstrated a biexponential pattern in which none of the chelators completely inhibited SPM-1. A homology model of SPM-1 was developed on the basis of the IMP-1 crystal structure, which showed the protein folding and active-site structure characteristic of metallo-β-lactamases and which provides an explanation for the kinetic profiles observed.

scholarly journals Genetic and biochemical characterization of the Na + /H + antiporters of Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Sara Foreman ◽  
Kristina Ferrara ◽  
Teri Hreha ◽  
Ana Duran-Pinedo ◽  
Jorge Frias-Lopez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biochemical Characterization ◽  
Physiological Role ◽  
Glycerol Metabolism ◽  
Kinetic Properties ◽  
Wild Type ◽  
Wide Range ◽  
Quadruple Mutant

Pseudomonas aeruginosa has four Na + /H + antiporters that interconvert and balance Na + and H + gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we have constructed four strains, each of which has only one antiporter: NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na + /H + antiporters. Although the antiporters of P. aeruginosa have previously been studied, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa . The strains expressing only NhaB or Mrp, the two electrogenic antiporters, are able to grow essentially as the wild type across a range of [Na + ] and pH. Strains with only NhaP or NhaP2, which are electroneutral, grow more poorly at increasing [Na + ], especially at high pH, with NhaP the most sensitive. The strain with no Na + /H + antiporters is extremely sensitive to [Na + ] and shows essentially no Na + (Li + )/H + antiporter activity but retains most K + /H + antiporter activity of the wild type at pH 7.5 and approximately half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. RNA-seq analysis of the quadruple deletion strain showed widespread changes, including pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological role of these proteins and their activities in P. aeruginosa . Importance Pseudomonas aeruginosa has four Na + /H + antiporters that connect and interconvert its Na + and H + gradients. We have constructed four deletion mutants, each of which has only one of the four Na + /H + antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH and [Na + ], whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH. We also constructed a quadruple mutant, lacking all four antiporters in which the H + and Na + gradients are disconnected. This will make it possible to study the role of the two gradients independently.

scholarly journals Evaluation of antimycobacterial rhamnolipid production from non-cytotoxic strains of Pseudomonas aeruginosa isolated from rhizospheric soil of medicinal plants

2016 ◽  
Vol 4 (2) ◽  
pp. 112 ◽  
Author(s):  
Alok K Mishra ◽  
Rikesh K Dubey ◽  
Shivraj M Yabaji ◽  
Swati Jaiswal
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Medicinal Plants ◽  
Biochemical Characterization ◽  
Rhizospheric Soil ◽  
Rhamnolipid Production ◽  
Medicinal Value ◽  
Wide Range ◽  
Rrna Sequencing ◽  
Inhibitory Potential ◽  
Potent Inhibitory Activity

Rhamnolipids (RLs) are the bacterial derived biosurfactants and known for a wide range of industrial and therapeutic applications. They exhibit potent anti-bacterial activity against various gram positive, gram negative and acid fast bacteria including Mycobacterium tuberculosis. Since, Pseudomonas is one of the largest known genuses containing a variety of rhamnolipid producing strains. Therefore, in this study, we selectively isolated the Pseudomonas aeruginosa strains from the rhizospheric soil of the Indian plants of medicinal value, e.g. Azadirachta Indica and Ficus spp., and evaluated them for their natural ability to produce antibacterial rhamnolipids. The bacteria were identified on the basis of 16s rRNA sequencing and biochemical characterization. Among 33 of P. aeruginosa isolates from different soil samples, four isolates showed potent inhibitory activity against methicillin resistant Staphylococcus aureus (MRSA) and fast grower mycobacterial spp. The inhibitory potential of the isolates was found to be correlated with their ability to produce RLs in the medium. The industrial viability of the strains was assessed on the basis of cytotoxicity determining alternative allele, exoS/exoU and cell mediated cytotoxicity against murine macrophages J774.1. The newly isolated strains harbor exoS allele and exhibits lower cell mediated cytotoxicity on macrophage cell line as compared to the clinical strains PA-BAA-427 and PA-27853 used as a control in this study.Evaluation of antimycobacterial rhamnolipid production from non-cytotoxic strains of Pseudomonas aeruginosa isolated from rhizospheric soil of medicinal plants

Growth and biochemical characterization of associations between cyanobionts and wheat seedlings in co-culturing experiments

Biologia ◽  
2011 ◽  
Vol 66 (1) ◽  
Author(s):  
Anjuli Sood ◽  
Pawan Singh ◽  
Arun Kumar ◽  
Rajendra Singh ◽  
Radha Prasanna
Keyword(s):  
Amino Acids ◽  
Indoleacetic Acid ◽  
Biochemical Characterization ◽  
Shoot Biomass ◽  
Wheat Seedlings ◽  
Symbiotic Associations ◽  
Plant Groups ◽  
Wide Range ◽  
Root And Shoot Biomass

AbstractN2-fixing cyanobacteria are unique in their capacity to form symbiotic associations with a wide range of eukaryotic hosts belonging to different plant groups. The present study was undertaken to analyze the interactions of the cyanobiont PI 01 (from Azolla pinnata) and Nostoc PCC 9229 (from Gunnera monoika) with wheat seedlings, in co-culturing experiments. Each of the cyanobionts enhanced significantly the volume of root and shoot biomass in the experimental cultures. The transverse sections of roots in the co-cultured seedlings revealed the presence of aseriate packets of cyanobionts below the root epidermis. The investigated cyanobionts excreted amino acids (His, Met, Val) and sugars into the medium, while indoleacetic acid was detected when the cyanobionts were grown in a tryptophan containing medium. During the co-culturing, sugars and proline were detected in the extracellular filtrates. It can be hypothesized that these sugars and amino acids may serve as signal substances in the development of functional associations between the relevant cyanobionts and the wheat seedlings.

scholarly journals Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-β-Lactamase Gene,blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia

2012 ◽  
Vol 56 (12) ◽  
pp. 6154-6159 ◽  
Author(s):  
Dongeun Yong ◽  
Mark A. Toleman ◽  
Jan Bell ◽  
Brett Ritchie ◽  
Rachael Pratt ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Southern Hybridization ◽  
Biochemical Characterization ◽  
Gene Bank ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Lactamase Gene ◽  
Genetic Context

ABSTRACTThree clinicalPseudomonas aeruginosaisolates (WCH2677, WCH2813, and WCH2837) isolated from the Women's and Children's Hospital, Adelaide, Australia, produced a metallo-β-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designatedblaAIM-1, was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCRelement, ISCR15. Southern hybridization studies indicated the movement of both ISCR15andblaAIM-1within the three different clinical isolates. AIM-1 hydrolyzes most β-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higherkcatvalues for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat seriousP. aeruginosaand other Gram-negative infections.

scholarly journals Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

2004 ◽  
Vol 381 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Jing WU ◽  
Mayur A. PATEL ◽  
Appavu K. SUNDARAM ◽  
Ronald W. WOODARD
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Kinetic Studies ◽  
Maximum Activity ◽  
Reading Frame ◽  
Sequential Mechanism ◽  
Phosphate Synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with Km=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and kcat=5.9 s−1 at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

scholarly journals Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Paola R. Beassoni ◽  
Lucas A. Gallarato ◽  
Cristhian Boetsch ◽  
Mónica N. Garrido ◽  
Angela T. Lisa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Active Site ◽  
Inorganic Phosphate ◽  
Biochemical Characterization ◽  
E Coli ◽  
Activity Dependent ◽  
Hydrolysis Of ◽  
Molecular Modeling And Docking

Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4+ is an activator of the enzyme and may function at concentrations lower than those of K+; (iii) Zn2+ is also an activator of paPpx and may substitute Mg2+ in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg2+ and capable of producing ATP regardless of the presence or absence of K+ or NH4+ ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity.

Plant ABC transporters: time for biochemistry?

2015 ◽  
Vol 43 (5) ◽  
pp. 931-936 ◽  
Author(s):  
François Lefèvre ◽  
Amandine Baijot ◽  
Marc Boutry
Keyword(s):  
Abc Transporters ◽  
Biochemical Characterization ◽  
Direct Methods ◽  
Membrane Transporters ◽  
Current Status ◽  
Isolated Cells ◽  
Abc Proteins ◽  
Physiological Processes ◽  
Wide Range

ATP-binding cassette (ABC) proteins form a large and ubiquitous family, most members of which are membrane-associated primary transporters. Plant genomes code for a particularly large number of these ABC proteins, with more than 120 genes present in both Arabidopsis thaliana and Oryza sativa (rice). Although plant ABC transporters were initially identified as detoxifiers, sequestering xenobitotics into the vacuole, they were later found to be involved in a wide range of essential physiological processes. Currently, the exact substrates transported by most of these transporters are still unknown and we therefore cannot exclude that a single substrate (e.g. a hormone) is responsible for the diversity of physiological roles. This gap in our knowledge is mainly due to the fact that only a few studies have used direct methods to identify the substrates of these membrane transporters. To address this issue, transport assays involving isolated cells, vesicular membranes or reconstituted liposomes are essential. In this review, we will highlight the importance of the direct biochemical characterization of plant ABC transporters and give some insights into the current status of the homologous and heterologous expression of such proteins.

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