Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae

2001 ◽  
Vol 355 (2) ◽  
pp. 431-435 ◽  
Author(s):  
Daniel R. SYLVESTER ◽  
Emilio ALVAREZ ◽  
Arun PATEL ◽  
Kapila RATNAM ◽  
Howard KALLENDER ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Substrate Inhibition ◽  
Coli Strain ◽  
Cell Protein ◽  
Binding Motif ◽  
Gram Positive ◽  
E Coli ◽  
Starting Point ◽  
Good Starting Point

The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.

scholarly journals Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae

2001 ◽  
Vol 355 (2) ◽  
pp. 431 ◽  
Author(s):  
Daniel R. SYLVESTER ◽  
Emilio ALVAREZ ◽  
Arun PATEL ◽  
Kapila RATNAM ◽  
Howard KALLENDER ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Gram Positive ◽  
Identification And Characterization

Functional characterization of the Haemophilus influenzae 4.5S RNA

1998 ◽  
Vol 44 (1) ◽  
pp. 91-94
Author(s):  
G Scott Jenkins ◽  
Mark S Chandler ◽  
Pamela S Fink
Keyword(s):  
Haemophilus Influenzae ◽  
Functional Characterization ◽  
Coli Strain ◽  
Northern Analysis ◽  
Gene Encoding ◽  
E Coli ◽  
Functional Homolog ◽  
Polymerase Chain ◽  
4.5S Rna

The putative 4.5S RNA of Haemophilus influenzae was identified in the genome by computer analysis, amplified by the polymerase chain reaction, and cloned. We have determined that this putative 4.5S RNA will complement an Escherichia coli strain conditionally defective in 4.5S RNA production. The predicted secondary structures of the molecules were quite similar, but Northern analysis showed that the H. influenzae RNA was slightly larger than the E. coli RNA. The H. influenzae gene encoding this RNA is the functional homolog of the ffs gene in E. coli. Key words: ffs gene, complementation studies, small RNA, prokaryotic genetics.

scholarly journals Structural and functional characterization of proteins from the fire blight pathogen Erwinia amylovora. A review on the state of the art

2020 ◽  
Author(s):  
Stefano Benini
Keyword(s):  
Fire Blight ◽  
Erwinia Amylovora ◽  
Functional Characterization ◽  
Data Bank ◽  
Knock Out ◽  
X Ray ◽  
X Ray Crystallography ◽  
Starting Point ◽  
Good Starting Point

Abstract Together with genome analysis and knock-out mutants, structural and functional characterization of proteins provide valuable hints on the biology of the organism under investigation. Structural characterization can be achieved by techniques such as X-ray crystallography, NMR, Cryo-EM. The information derived from the structure are a good starting point to comprehend the details of the proteins molecular function for a better understanding of their biological role. This review aims at describing the progress in the structural and functional characterization of proteins from the plant pathogen Erwinia amylovora obtained by structural biology and currently deposited in the Protein Data Bank.

scholarly journals Purification and characterization of Haemophilus influenzae pili, and their structural and serological relatedness to Escherichia coli P and mannose-sensitive pili.

1985 ◽  
Vol 161 (1) ◽  
pp. 145-159 ◽  
Author(s):  
N G Guerina ◽  
S Langermann ◽  
G K Schoolnik ◽  
T W Kessler ◽  
D A Goldmann
Keyword(s):  
Haemophilus Influenzae ◽  
Cross Reactivity ◽  
Coli Strain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Cell Agglutination ◽  
E Coli ◽  
Amino Terminal ◽  
Multiple Copies

Haemophilus influenzae pili were purified, and their physical and serological properties were examined. The solution properties of the pili were determined, and then a purification scheme involving repeated cycles of precipitation and solubilization was developed. The purified pili from one type b isolate (A02) were found to consist of multiple copies of a 25,000 mol wt subunit. Amino-terminal sequence analysis of A02 pili was carried out to 40 amino acid residues, and a remarkable degree of sequence homology was found with E. coli P and mannose-sensitive (MS) pili (27.5 and 25% homology, respectively). Purified A02 pili were found to be highly immunogenic, and serological analysis by enzyme-linked immunosorbent assay and whole piliated cell agglutination revealed significant cross-reactivity between A02 pilus antiserum and the pili of seven other H. influenzae strains tested (heterologous titers = 2-100% of the homologous titer). Cross-reactivity was also observed between the H. influenzae pili (five of eight strains tested) and the P pili from E. coli strains HU849 and 3669; no cross-reactivity was detected with MS pili from E. coli strain H10407 and C94. The structural similarities between H. influenzae and E. coli P and MS pili suggest a common gene ancestry.

scholarly journals Genomic and Proteomic Characterization of the Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Strain CCUG 73778: A Virulent, Nosocomial Outbreak Strain

2020 ◽  
Vol 8 (6) ◽  
pp. 893 ◽  
Author(s):  
Daniel Jaén-Luchoro ◽  
Antonio Busquets ◽  
Roger Karlsson ◽  
Francisco Salvà-Serra ◽  
Christina Åhrén ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Coli Strain ◽  
University Hospital ◽  
Hospital Environment ◽  
Resistance Factors ◽  
E Coli ◽  
Extended Spectrum ◽  
Liquid Chromatography Tandem Mass

Escherichia coli strain CCUG 78773 is a virulent extended-spectrum β-lactamase (ESBL)-producing ST131-O25b type strain isolated during an outbreak at a regional university hospital. The complete and closed genome sequence, comprising one chromosome (5,076,638 bp) and six plasmids (1718–161,372 bp), is presented. Characterization of the genomic features detected the presence of 59 potential antibiotic resistance factors, including three prevalent β-lactamases. Several virulence associated elements were determined, mainly related with adherence, invasion, biofilm formation and antiphagocytosis. Twenty-eight putative type II toxin-antitoxin systems were found. The plasmids were characterized, through in silico analyses, confirming the two β-lactamase-encoding plasmids to be conjugative, while the remaining plasmids were mobilizable. BLAST analysis of the plasmid sequences showed high similarity with plasmids in E. coli from around the world. Expression of many of the described virulence and AMR factors was confirmed by proteomic analyses, using bottom-up, liquid chromatography-tandem mass spectrometry (LC-MS/MS). The detailed characterization of E. coli strain CCUG 78773 provides a reference for the relevance of genetic elements, as well as the characterization of antibiotic resistance and the spread of bacteria harboring ESBL genes in the hospital environment.

scholarly journals Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

2008 ◽  
Vol 51 (3) ◽  
pp. 473-482 ◽  
Author(s):  
Dorismey Vieira Tokano ◽  
Marisa Emiko Kawaichi ◽  
Emerson José Venâncio ◽  
Marilda Carlos Vidotto
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Iron Uptake ◽  
Expression Vector ◽  
Iron Acquisition ◽  
Coli Strain ◽  
High Yield ◽  
High Similarity ◽  
E Coli

The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.

scholarly journals Characterization of Escherichia coliD-arabinose 5-phosphate isomerase encoded by kpsF: implications for group 2 capsule biosynthesis

2006 ◽  
Vol 395 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Timothy C. Meredith ◽  
Ronald W. Woodard
Keyword(s):  
Biochemical Properties ◽  
Coli Strain ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Catalytic Constant ◽  
Group 2 ◽  
Strain Cft073 ◽  
K Antigen ◽  
Pentose Pathway

In Escherichia coli, there are multiple paralogous copies of the enzyme API [A5P (D-arabinose 5-phosphate) isomerase], which catalyses the conversion of the pentose pathway intermediate Ru5P (D-ribulose 5-phosphate) into A5P. A5P is a precursor of Kdo (3-deoxy-D-manno-octulosonate), an integral carbohydrate component of various glycolipids coating the surface of the OM (outer membrane) of Gram-negative bacteria, including LPS (lipopolysaccharide) and many group 2 K-antigen capsules. The K-antigen-specific API KpsF has been cloned from the uropathogenic E. coli strain CFT073 and its biochemical properties characterized. Purified recombinant KpsF [K-API (K-antigen API)] is tetrameric and has optimal activity at pH 7.8. The enzyme is specific for A5P and Ru5P, with Km (app) values of 0.57 mM for A5P and 0.3 mM for Ru5P. The apparent kcat in the A5P to Ru5P direction is 15 and 19 s−1 in the Ru5P to A5P direction. While most of the properties are quite similar to its LPS API counterpart KdsD, the catalytic constant is nearly 10-fold lower. K-API is now the second Kdo biosynthetic related gene that has been characterized from the kps group 2 capsule cluster.

scholarly journals Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

2004 ◽  
Vol 186 (8) ◽  
pp. 2346-2354 ◽  
Author(s):  
Karen O'Dwyer ◽  
Joseph M. Watts ◽  
Sanjoy Biswas ◽  
Jennifer Ambrad ◽  
Michael Barber ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Kinetic Studies ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
E Coli ◽  
Trna Species ◽  
Sequential Mechanism ◽  
Trna Methyltransferase ◽  
Gel Mobility Shift

ABSTRACT Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{CAG}}^{\mathit{Leu}}\) \end{document} . Other heterologous nonsubstrate tRNA species, like \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{GGT}}^{\mathit{Thr}}\) \end{document} , tRNAPhe, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{TGC}}^{\mathit{Ala}}\) \end{document} , bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).

scholarly journals What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin

2019 ◽  
Vol 7 (2) ◽  
pp. 59 ◽  
Author(s):  
Marcus Krüger ◽  
Peter Richter ◽  
Sebastian Strauch ◽  
Adeel Nasir ◽  
Andreas Burkovski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
High Sensitivity ◽  
Coli Strain ◽  
Photodynamic Treatment ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Bacterial Model

Due to the increasing development of antibiotic resistances in recent years, scientists search intensely for new methods to control bacteria. Photodynamic treatment with porphyrins such as chlorophyll derivatives is one of the most promising methods to handle bacterial infestation, but their use is dependent on illumination and they seem to be more effective against Gram-positive bacteria than against Gram-negatives. In this study, we tested chlorophyllin against three bacterial model strains, the Gram-positive Bacillus subtilis 168, the Gram-negative Escherichia coli DH5α and E. coli strain NR698 which has a deficient outer membrane, simulating a Gram-negative “without” its outer membrane. Illuminated with a standardized light intensity of 12 mW/cm2, B. subtilis showed high sensitivity already at low chlorophyllin concentrations (≤105 cfu/mL: ≤0.1 mg/L, 106–108 cfu/mL: 0.5 mg/L), whereas E. coli DH5α was less sensitive (≤105 cfu/mL: 2.5 mg/L, 106 cfu/mL: 5 mg/L, 107–108 cfu/mL: ineffective at ≤25 mg/L chlorophyllin). E. coli NR698 was almost as sensitive as B. subtilis against chlorophyllin, pointing out that the outer membrane plays a significant role in protection against photodynamic chlorophyllin impacts. Interestingly, E. coli NR698 and B. subtilis can also be inactivated by chlorophyllin in darkness, indicating a second, light-independent mode of action. Thus, chlorophyllin seems to be more than a photosensitizer, and a promising substance for the control of bacteria, which deserves further investigation.

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