Metal binding by the peptidoglycan sacculus of Escherichia coli K-12

1984 ◽  
Vol 30 (2) ◽  
pp. 204-211 ◽  
Author(s):  
B. D. Hoyle ◽  
T. J. Beveridge
Keyword(s):  
Escherichia Coli ◽  
Aqueous Solution ◽  
Bacillus Subtilis ◽  
Metal Binding ◽  
Sodium Dodecyl ◽  
Dry Weight ◽  
Metallic Ions ◽  
Trypsin Treatment ◽  
Whole Cells ◽  
K 12

The peptidoglycan of Escherichia coli K-12 strain AB264 was isolated by treating whole cells with sodium dodecyl sulfate and was purified by deoxyribonuclease, ribonuclease, and trypsin treatment. Like the peptidoglycan of Bacillus subtilis, this peptidoglycan proved able to bind substantial amounts of metallic ions from aqueous solution. In particular, most metals of the transition I series were bound from solution in amounts ≥ 1 μmol/mg dry weight peptidoglycan.

scholarly journals Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

2017 ◽  
Vol 3 (5) ◽  
pp. 139
Author(s):  
Mariana Wahjudi ◽  
Catherina . ◽  
Nita Marcelia Wangunhardjo ◽  
Ernest Suryadjaja ◽  
Xavier Daniel
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Recombinant Plasmid ◽  
Sodium Dodecyl ◽  
Xylanase Activity ◽  
Cellular Protein ◽  
Host Cells ◽  
Chromosomal Dna ◽  
Clear Zone ◽  
Sds Page

<p class="Els-Abstract-text">The <em>xyn</em>B gene of <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the <em>xyn</em>B gene from <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the <em>xyn</em>B gene in <em>Escherichia coli </em>Origami as host cells. The <em>x</em><em>yn</em>B gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The <em>xyn</em>B gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for <em>xyn</em>B gene and for the vector, then continued by restriction analyses.  The result showed that transformants clone 9 and 10 bear the recombinant pMMB-<em>xyn</em>B plasmid. The xylanase activity of <em>xyn</em>B gene in <em>Escherichia coli</em> Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-<em>xyn</em>B recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the <em>xyn</em>B gene of <em>Bacillus subtilis</em> subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the <em>Escherichia coli</em> Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.</p>

Isolation and purification of a Campylobacter upsaliensis autolysin

1999 ◽  
Vol 45 (1) ◽  
pp. 23-30
Author(s):  
Somchai Santiwatanakul ◽  
Noel R Krieg
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Micrococcus Luteus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Isolation And Purification ◽  
Whole Cells ◽  
Sds Page ◽  
Autolytic Activity ◽  
Campylobacter Upsaliensis

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS) - PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.Key words: autolysin, Campylobacter upsaliensis, zymogram, murein hydrolase.

scholarly journals Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2341-2351 ◽  
Author(s):  
Mario Juhas ◽  
Daniel R. Reuß ◽  
Bingyao Zhu ◽  
Fabian M. Commichau
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Essential Gene ◽  
Building Blocks ◽  
Essential Genes ◽  
Minimal Cell ◽  
E Coli ◽  
Cell Factories ◽  
Gene Sets ◽  
K 12

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell ‘chassis’. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

1986 ◽  
Vol 32 (7) ◽  
pp. 594-601 ◽  
Author(s):  
F. G. Ferris ◽  
T. J. Beveridge
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Metal Binding ◽  
Metal Salt ◽  
Binding Capacity ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Metallic Ions ◽  
Intact Cells ◽  
K 12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0–7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran–polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.

scholarly journals Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

2006 ◽  
Vol 74 (4) ◽  
pp. 2233-2244 ◽  
Author(s):  
Alison S. Low ◽  
Francis Dziva ◽  
Alfredo G. Torres ◽  
Jessenya L. Martinez ◽  
Tracy Rosser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
Induced Expression ◽  
E Coli ◽  
Extracellular Matrix Components ◽  
K 12

ABSTRACT Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

1980 ◽  
Vol 29 (2) ◽  
pp. 704-713 ◽  
Author(s):  
Henk Gankema ◽  
Jan Wensink ◽  
Pieter A. M. Guinée ◽  
Wim H. Jansen ◽  
Bernard Witholt
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Sucrose Density Gradient ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Freeze Fracture Electron Microscopy ◽  
K 12

The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers.

scholarly journals Ubiquinone biosynthesis in Escherichia coli K-12. Accumulation of an octaprenol, farnesylfarnesylgeraniol, by a multiple aromatic auxotroph

1971 ◽  
Vol 123 (3) ◽  
pp. 435-443 ◽  
Author(s):  
J. A. Hamilton ◽  
G. B. Cox
Keyword(s):  
Escherichia Coli ◽  
Mass Spectroscopy ◽  
Active Form ◽  
Side Chain ◽  
Whole Cells ◽  
Cell Extracts ◽  
K 12

Cell extracts of a multiple aromatic auxotroph of Escherichia coli K-12, strain AB2830, grown in the absence of precursors of the quinone rings of the ubiquinone and menaquinone molecules, converted 4-hydroxy[U-14C]benzoate into a mixture of 3-octaprenyl-4-hydroxybenzoate and 2-octaprenylphenol. An octaprenol, farnesylfarnesylgeraniol, was isolated from such cell extracts and characterized by n.m.r. and mass spectroscopy. Neither the octaprenol, nor polyprenylation of 4-hydroxy[U-14C]benzoate, could be detected in cell extracts of strain AB2830 grown in the presence of 0.1mm-4-hydroxybenzoate. It was concluded that, in the biosynthesis of ubiquinone, the polyprenyl side chain is added to 4-hydroxybenzoate as a C40 unit, the active form of which is converted by cell extracts into farnesylfarnesylgeraniol. The multiple aromatic auxotroph, when grown in the absence of 4-hydroxybenzoate but in the presence of 4-aminobenzoate, converted the latter compound into 3-octaprenyl-4-aminobenzoate. This compound was isolated from whole cells and characterized by n.m.r. and mass spectroscopy.

scholarly journals Removal of the antimicrobial activity from fortified effluents with fluoroquinolones by photocatalytic processes: a comparative study of differently synthesized TiO2-N

2020 ◽  
Author(s):  
Wilson Augusto Lima Venancio ◽  
Caio Rodrigues-Silva ◽  
Mylena Spina ◽  
José Roberto Guimarães
Keyword(s):  
Escherichia Coli ◽  
Aqueous Solution ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Photocatalytic Oxidation ◽  
Sol Gel ◽  
Titanium Isopropoxide ◽  
Reaction Intermediates ◽  
Titanium Butoxide ◽  
Photocatalytic Processes

Abstract This study presents a comparison of three methods for TiO2-N synthesis that were applied in the photocatalytic oxidation of the fluoroquinolones (FQs) ciprofloxacin, ofloxacin, and lomefloxacin in aqueous solution. The TiO2-N bandgap is small enough to allow the use of solar energy in the photocatalytic oxidation (PCO) reactions. The TiO2 doped by a sol-gel method with titanium butoxide (TiO2–N–BUT) and titanium isopropoxide (TiO2–N–PROP) as the precursor were effective as the TiO2 (P25) impregnation with urea (TiO2-N-P25) to degrade the FQs. The FQ degradation was higher by 74, 65, and 91%, respectively for TiO2–N–BUT, TiO2–N–PROP, and TiO2-N (load 50 mg L−1, 20 min of reaction under 28 W UV-ASolar). The TiO2-P25 with urea showed the best performance in FQ degradation. The reaction intermediates might present modifications in their acceptor groups by PCO and, because of that the antimicrobial activity dropped as the reaction time increased. Reactions with TiO2-N-P25 (100 mg L−1) and TiO2-N-BUT (100 mg L−1) achieved ≥ 80% of antimicrobial activity removal from the mixed FQ solution (Cciprofloxacin = 100 μg L−1; Cofloxacin = 100 μg L−1; Clomefloxacin = 100 μg L−1) after 40 min of reaction, for both for Escherichia coli and Bacillus subtilis.

scholarly journals Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

2015 ◽  
Vol 81 (17) ◽  
pp. 5900-5906 ◽  
Author(s):  
Yoshihiro Ojima ◽  
Minh Hong Nguyen ◽  
Reiki Yajima ◽  
Masahito Taya
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Laser Scanning Microscopy ◽  
Content Type ◽  
E Coli ◽  
Enhanced Production ◽  
K 12

ABSTRACTMicrobial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation ofEscherichia colicells by overexpression of thebcsBgene was investigated. The flocculation induced by overexpression ofbcsBwas consistent among the variousE. colistrains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeledE. colicells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) inE. coliflocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore,bcsB-inducedE. coliflocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbAand ΔdsbBstrains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production ofE. colicells.

Penicillin tolerance in Neisseria gonorrhoeae: evidence disallowing a penicillinase-mediated mechanism from a refined microbiological assay method

1976 ◽  
Vol 22 (1) ◽  
pp. 76-82 ◽  
Author(s):  
R. A. Scudamore ◽  
M. Goldner
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Dry Weight ◽  
Microbiological Assay ◽  
Assay Method ◽  
Benzyl Penicillin ◽  
Quantitative Evidence ◽  
Quantitative Manner ◽  
K 12

A microbiological assay method has been developed and applied to Neisseria gonorrhoeae, for the purpose of detecting enzymatic deactivation of benzyl penicillin. Calibration of the method, using strains of Escherichia coli K-12 with previously reported penicillinase (EC 3.5.2.6.) activities, has shown that it is extremely sensitive and may be used in a quantitative manner. At the limit of sensitivity the test is able to detect penicillin breakdown in the order of 3 × 10−3 μg in 48 h, which is equivalent to about 7 × 10−8 μmol/min per milligram dry weight of cells. Over 100 strains of N. gonorrhoeae, most of them resistant to penicillin, were screened for their ability to deactivate penicillin during 48 h of growth in the presence of subinhibitory levels. No deactivation was detected. It is concluded, from quantitative evidence, that reduced penicillin sensitivity in N. gonorrhoeae is not due to the enzymatic deactivation of the antibiotic.

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