scholarly journals P1 Trisaccharide (Galα1,4Galβ1,4GlcNAc) Synthesis by Enzyme Glycosylation Reactions Using Recombinant Escherichia coli

2003 ◽  
Vol 69 (4) ◽  
pp. 2110-2115 ◽  
Author(s):  
Ziye Liu ◽  
Yuquan Lu ◽  
Jianbo Zhang ◽  
Keith Pardee ◽  
Peng George Wang
Keyword(s):  
Escherichia Coli ◽  
Sucrose Synthase ◽  
Pathogenic Bacteria ◽  
Enzymatic Reaction ◽  
Genetically Engineered ◽  
Reaction Volume ◽  
Recombinant Bacteria ◽  
Preparative Scale ◽  
E Coli ◽  
Oligosaccharide Moiety

ABSTRACT The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Galα1,4Galβ1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori β-l,4-galactosyltransferase and a Neisseria meningitidis α-1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli

2020 ◽  
Vol 14 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Maryam Ahankoub ◽  
Gashtasb Mardani ◽  
Payam Ghasemi-Dehkordi ◽  
Ameneh Mehri-Ghahfarrokhi ◽  
Abbas Doosti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Performance ◽  
Human Cancer ◽  
Genetically Engineered ◽  
Hazardous Substances ◽  
E Coli ◽  
Spiked Soil ◽  
Engineered Escherichia Coli ◽  
Genetically Manipulated ◽  
Hplc Measurement

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

scholarly journals High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials

2020 ◽  
Vol 367 (22) ◽  
Author(s):  
Chris Coward ◽  
Gopujara Dharmalingham ◽  
Omar Abdulle ◽  
Tim Avis ◽  
Stephan Beisken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Selective Advantage ◽  
Mechanisms Of Action ◽  
Over Expression ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Established Technique ◽  
Bacterial Transposon

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

PCR for the Specific Detection of an Escherichia coli O157:H7 Laboratory Control Strain

2015 ◽  
Vol 78 (9) ◽  
pp. 1738-1744 ◽  
Author(s):  
MICHAEL KNOWLES ◽  
DOMINIC LAMBERT ◽  
GEORGE HUSZCZYNSKI ◽  
MARTINE GAUTHIER ◽  
BURTON W. BLAIS
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Control Measure ◽  
Positive Control ◽  
Food Microbiology ◽  
Escherichia Coli O157 ◽  
Control Strain ◽  
E Coli ◽  
Signature Sequences ◽  
Materials Used

Control strains of bacterial pathogens such as Escherichia coli O157:H7 are commonly processed in parallel with test samples in food microbiology laboratories as a quality control measure to assure the satisfactory performance of materials used in the analytical procedure. Before positive findings can be reported for risk management purposes, analysts must have a means of verifying that pathogenic bacteria (e.g., E. coli O157:H7) recovered from test samples are not due to inadvertent contamination with the control strain routinely handled in the laboratory environment. Here, we report on the application of an in-house bioinformatic pipeline for the identification of unique genomic signature sequences in the development of specific oligonucleotide primers enabling the identification of a common positive control strain, E. coli O157:H7 (ATCC 35150), using a simple PCR procedure.

Mannose-Binding Activity of Escherichia coli: a Determinant of Attachment and Ingestion of the Bacteria by Macrophages

1980 ◽  
Vol 29 (2) ◽  
pp. 417-424
Author(s):  
Zvi Bar-Shavit ◽  
Rachel Goldman ◽  
Itzhak Ofek ◽  
Nathan Sharon ◽  
David Mirelman
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Pathogenic Bacteria ◽  
Binding Activity ◽  
Exponential Phase ◽  
Restrictive Temperature ◽  
Filamentous Bacteria ◽  
Phagocytic Cells ◽  
E Coli ◽  
Mannose Binding

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [ 14 C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α- d -mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters ( R = 0.98, P < 0.001). The internalization of the filamentous cells attached to macrophages during 45 min of incubation was much less efficient (20%) compared to that of exponential-phase, stationary-phase, or antibody-coated filamentous bacteria (90%). The results indicate that the mannose-binding activity of E. coli determines the recognition of the organisms by phagocytes. They further suggest that administration of β-lactam antibiotics may impair elimination of certain pathogenic bacteria by inducing the formation of filaments which are inefficiently internalized by the host's phagocytic cells.

scholarly journals Escherichia coli as a genetic tool

1985 ◽  
Vol 95 (3) ◽  
pp. 611-618
Author(s):  
Naomi Datta
Keyword(s):  
Escherichia Coli ◽  
Academic Research ◽  
Genetically Engineered ◽  
Cloning And Expression ◽  
Biological Research ◽  
New Techniques ◽  
E Coli ◽  
Genetic Tool ◽  
The Past ◽  
Made In

SUMMARYThe study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.

scholarly journals Detection and Quantification of Superoxide Formed within the Periplasm of Escherichia coli

2006 ◽  
Vol 188 (17) ◽  
pp. 6326-6334 ◽  
Author(s):  
Sergei Korshunov ◽  
James A. Imlay
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Phagocytic Cells ◽  
Free Living ◽  
E Coli ◽  
Superoxide Formation ◽  
Genes Encoding ◽  
Copper Zinc ◽  
Primary Substrate

ABSTRACT Many gram-negative bacteria harbor a copper/zinc-containing superoxide dismutase (CuZnSOD) in their periplasms. In pathogenic bacteria, one role of this enzyme may be to protect periplasmic biomolecules from superoxide that is released by host phagocytic cells. However, the enzyme is also present in many nonpathogens and/or free-living bacteria, including Escherichia coli. In this study we were able to detect superoxide being released into the medium from growing cultures of E. coli. Exponential-phase cells do not normally synthesize CuZnSOD, which is specifically induced in stationary phase. However, the engineered expression of CuZnSOD in growing cells eliminated superoxide release, confirming that this superoxide was formed within the periplasm. The rate of periplasmic superoxide production was surprisingly high and approximated the estimated rate of cytoplasmic superoxide formation when both were normalized to the volume of the compartment. The rate increased in proportion to oxygen concentration, suggesting that the superoxide is generated by the adventitious oxidation of an electron carrier. Mutations that eliminated menaquinone synthesis eradicated the superoxide formation, while mutations in genes encoding respiratory complexes affected it only insofar as they are likely to affect the redox state of menaquinone. We infer that the adventitious autoxidation of dihydromenaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm of E. coli. This endogenous superoxide may create oxidative stress in that compartment and be a primary substrate of CuZnSOD.

scholarly journals Lactic Acid Bacteria (Lactobacillus bulgaricus and Streptococcus thermophillus) in Yogurt Inhibit the Growth of Escherichia coli, Salmonella typhimurium, and Shigella sp. In Vitro

2021 ◽  
Vol 31 (4) ◽  
pp. 2
Author(s):  
IDSAP Peramiarti
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Salmonella Typhimurium ◽  
Pathogenic Bacteria ◽  
Test Method ◽  
P Value ◽  
E Coli ◽  
Significant Difference

Diarrhea is defecation with a frequency more often than usual (three times or more) a day (10 mL/kg/day) with a soft or liquid consistency, even in the form of water alone. Pathogenic bacteria, such as Escherichia coli, Salmonella typhimurium, and Shigella sp., play a role in many cases, to which antibiotics are prescribed as the first-line therapy. However, since antibiotic resistance cases are often found, preventive therapies are needed, such as consuming yogurt, which is produced through a fermentation process by lactic acid bacteria (LAB). This research aimed to determine the activity of lactic acid bacteria (Liactobacillus bulgaricus and Streptococcus thermophilus) in yogurt in inhibiting the growth of the pathogenic bacteria E. coli, S. typhimurium, and Shigella sp. The research applied in vitro with the liquid dilution test method and the true experimental design research method with post-test-only and control group design. The design was used to see the inhibitory effect of yogurt LAB on the growth of E. coli, S. typhimurium, and Shigell sp. to compare the effect of several different yogurt concentrations, namely 20%, 40%, 60%, and 80%. The results of the Least Significance Different analysis showed that there was a significant difference between yogurt with a concentration of 0% and that with various concentrations in inhibiting the growth of E. coli, S. typhimurium, and Shigella sp. with a p-value of &lt;0.05. Whereas, there was no significant difference in the various concentrations of yogurt in inhibiting the growth of the three kinds of bacteria with a p-value of &gt; 0.05.<p class="Default" align="center"> </p>

scholarly journals A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2021 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Multiple Alternative ◽  
Z Ring ◽  
Cell Division Inhibitor

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism. Importance: Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

scholarly journals d-Serine induces distinct transcriptomes in diverse Escherichia coli pathotypes

Microbiology ◽  
2021 ◽  
Vol 167 (10) ◽  
Author(s):  
James P. R. Connolly ◽  
Natasha C. A. Turner ◽  
Jennifer C. Hallam ◽  
Patricia T. Rimbi ◽  
Tom Flett ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Species ◽  
Pathogenic Bacteria ◽  
Neonatal Meningitis ◽  
Published Data ◽  
Toxic Metabolite ◽  
Transcriptional Responses ◽  
Content Type ◽  
E Coli ◽  
Link Type

Appropriate interpretation of environmental signals facilitates niche specificity in pathogenic bacteria. However, the responses of niche-specific pathogens to common host signals are poorly understood. d-Serine (d-ser) is a toxic metabolite present in highly variable concentrations at different colonization sites within the human host that we previously found is capable of inducing changes in gene expression. In this study, we made the striking observation that the global transcriptional response of three Escherichia coli pathotypes – enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis-associated E. coli (NMEC) – to d-ser was highly distinct. In fact, we identified no single differentially expressed gene common to all three strains. We observed the induction of ribosome-associated genes in extraintestinal pathogens UPEC and NMEC only, and the induction of purine metabolism genes in gut-restricted EHEC, and UPEC indicating distinct transcriptional responses to a common signal. UPEC and NMEC encode dsdCXA – a genetic locus required for detoxification and hence normal growth in the presence of d-ser. Specific transcriptional responses were induced in strains accumulating d-ser (WT EHEC and UPEC/NMEC mutants lacking the d-ser-responsive transcriptional activator DsdC), corroborating the notion that d-ser is an unfavourable metabolite if not metabolized. Importantly, many of the UPEC-associated transcriptome alterations correlate with published data on the urinary transcriptome, supporting the hypothesis that d-ser sensing forms a key part of urinary niche adaptation in this pathotype. Collectively, our results demonstrate distinct pleiotropic responses to a common metabolite in diverse E. coli pathotypes, with important implications for niche selectivity.

Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli

2021 ◽  
Vol 118 (50) ◽  
pp. e2114013118
Author(s):  
Tamara N. Grund ◽  
Melanie Radloff ◽  
Di Wu ◽  
Hojjat G. Goojani ◽  
Luca F. Witte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Reduction Rate ◽  
Structural Diversity ◽  
Multidrug Resistant ◽  
E Coli ◽  
Structural And Functional Properties ◽  
Oxygen Reductase ◽  
General Architecture ◽  
Structural Insights

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I. Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

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