Displaying publications 1 - 20 of 40 in total

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  1. Sultana A, Tiash S
    J Control Release, 2021 04 10;332:233-244.
    PMID: 33561481 DOI: 10.1016/j.jconrel.2021.02.004
    E. coli mediated gene delivery faces a major drawback of low efficiency despite of being a safer alternative to viral vectors. This study showed a novel, simple and effective strategy to enhance invasive E. coli DH10B vector's efficiency in human epithelial cells. The bactofection efficiency of invasive E .coli vector was analyzed in nine cell lines. It demonstrated highest (16%) reporter gene (GFP) expression in cervical cells. Methods were employed to further enhance its efficiency by adding transfection reagents (trans-bactofection method) to promote entry into host cells, lysosomotropic reagents for escape from lysosomal degradation or antibiotics to lyse internalized bacteria. Increased bacterial entry, as elucidated from nil to 3% expression in liver cells, was obtained upon complexing bacteria with PULSin. Chloroquine mediated endosomal escape resulted in 7.2 folds increase whereas tetracycline addition to lyse internalized bacteria caused ≈90% of GFP in HeLa. Eventually, the combined effect of these three methods exhibited close to 100% GFP in cervical and remarkable increase of 138 folds in breast cells. This is the first study showing comparative study of vector's gene delivery ability in various epithelial cells of the human body with improving its delivery efficiency. These data demonstrated the potential of developed bactofection method to boost up the efficiency of other bacterial vectors also, which could further be used for effectual therapeutic gene delivery in human cells.
    Matched MeSH terms: Escherichia coli Proteins*
  2. Yu CY, Ang GY, Chong TM, Chin PS, Ngeow YF, Yin WF, et al.
    J Antimicrob Chemother, 2017 04 01;72(4):1253-1255.
    PMID: 28031273 DOI: 10.1093/jac/dkw541
    Matched MeSH terms: Escherichia coli Proteins/genetics*
  3. Mohd Yusoff MZ, Hashiguchi Y, Maeda T, Wood TK
    Biochem Biophys Res Commun, 2013 Oct 4;439(4):576-9.
    PMID: 24025676 DOI: 10.1016/j.bbrc.2013.09.016
    Pseudogenes are considered to be nonfunctional genes that lack a physiological role. By screening 3985 Escherichia coli mutants using chemochromic membranes, we found four pseudogenes involved in hydrogen metabolism. Knockouts of pseudogenes ydfW and ypdJ had a defective hydrogen phenotype on glucose and formate, respectively. Also, the knockout of pseudogene yqiG formed hydrogen from formate but not from glucose. For the yqiG mutant, 100% hydrogen recovery was obtained by the complementation of YqiG via a plasmid. The knockout of pseudogene ylcE showed hydrogen deficiency in minimal media which suggested that the role of YlcE is associated with cell growth. Hence, the products of these four pseudogenes play an important physiological role in hydrogen production in E. coli.
    Matched MeSH terms: Escherichia coli Proteins/genetics*; Escherichia coli Proteins/metabolism
  4. Mienda BS, Shamsir MS, Md Illias R
    J Biomol Struct Dyn, 2016 Aug;34(8):1705-16.
    PMID: 26513379 DOI: 10.1080/07391102.2015.1090341
    Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.
    Matched MeSH terms: Escherichia coli Proteins/genetics*; Escherichia coli Proteins/metabolism*
  5. Ho WS, Ou HY, Yeo CC, Thong KL
    BMC Genomics, 2015;16:199.
    PMID: 25879448 DOI: 10.1186/s12864-015-1421-8
    Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands.
    Matched MeSH terms: Escherichia coli Proteins/classification; Escherichia coli Proteins/genetics*
  6. Asi AM, Rahman NA, Merican AF
    J Mol Graph Model, 2004 Mar;22(4):249-62.
    PMID: 15177077
    Protein-ligand binding free energy values of wild-type and mutant C-terminal domain of Escherichia coli arginine repressor (ArgRc) protein systems bound to L-arginine or L-citrulline molecules were calculated using the linear interaction energy (LIE) method by molecular dynamics (MD) simulation. The binding behaviour predicted by the dissociation constant (K(d)) calculations from the binding free energy values showed preferences for binding of L-arginine to the wild-type ArgRc but not to the mutant ArgRc(D128N). On the other hand, L-citrulline do not favour binding to wild-type ArgRc but prefer binding to mutant ArgRc(D128N). The dissociation constant for the wild-type ArgRc-L-arginine complex obtained in this study is in agreement with reported experimental results. Our results also support the experimental data for the binding of L-citrulline to the mutant ArgRc(D128N). These showed that LIE method for protein-ligand binding free energy calculation could be applied to the wild-type and the mutant E. coli ArgRc-L-arginine and ArgRc-L-citrulline protein-ligand complexes and possibly to other transcriptional repressor-co-repressor systems as well.
    Matched MeSH terms: Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism*
  7. Afiqah RN, Paital B, Kumar S, Majeed AB, Tripathy M
    J. Mol. Recognit., 2016 11;29(11):544-554.
    PMID: 27406464 DOI: 10.1002/jmr.2554
    The inhibitory role of AgNO3 on glucose-mediated respiration in Escherichia coli has been investigated as a function of pH and temperature using Clark-type electrode, environmental scanning electron microscopy, and computational tools. In the given concentration of bacterial suspension (1 × 10(8)  CFU/ml), E. coli showed an increasing nonlinear trend of tetra-phasic respiration between 1-133 μM glucose concentration within 20 min. The glucose concentrations above 133 μM did not result any linear increment in respiration but rather showed a partial inhibition at higher glucose concentrations (266-1066 μM). In the presence of glucose, AgNO3 caused a concentration-dependent (47-1960 μM) inhibition of the respiration rate within 4 min of its addition. The respiration rate was the highest at pH 7-8 and then was decreased on either side of this pH range. The inhibitory action of AgNO3 upon bacterial respiration was the highest at 37 °C. The observations of the respiration data were well supported by the altered bacterial morphology as observed in electron microscopic study. Docking study indicated the AgNO3 binding to different amino acids of all respiratory complex enzymes in E. coli and thereby explaining its interference with the respiratory chain. Copyright © 2016 John Wiley & Sons, Ltd.
    Matched MeSH terms: Escherichia coli Proteins/metabolism; Escherichia coli Proteins/chemistry
  8. Wan Makhtar WR, Mohd Azlan M, Hassan NH, Aziah I, Samsurizal NH, Yusof NY
    Microbiol Resour Announc, 2020 Aug 13;9(33).
    PMID: 32817162 DOI: 10.1128/MRA.01497-19
    We describe here the draft genome sequence and basic characteristics of Escherichia coli isolate INF13/18/A, which was isolated from Universiti Sains Malaysia (USM) Hospital. This isolate was identified as an extended-spectrum β-lactamase-producing Escherichia coli strain harboring the antimicrobial resistance genes TEM, CTX-M-1, and CTX-M-9.
    Matched MeSH terms: Escherichia coli Proteins
  9. Goh KGK, Phan MD, Forde BM, Chong TM, Yin WF, Chan KG, et al.
    mBio, 2017 10 24;8(5).
    PMID: 29066548 DOI: 10.1128/mBio.01558-17
    Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.
    Matched MeSH terms: Escherichia coli Proteins/genetics*; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism
  10. Cheng KK, Lee BS, Masuda T, Ito T, Ikeda K, Hirayama A, et al.
    Nat Commun, 2014;5:3233.
    PMID: 24481126 DOI: 10.1038/ncomms4233
    Comparative whole-genome sequencing enables the identification of specific mutations during adaptation of bacteria to new environments and allelic replacement can establish their causality. However, the mechanisms of action are hard to decipher and little has been achieved for epistatic mutations, especially at the metabolic level. Here we show that a strain of Escherichia coli carrying mutations in the rpoC and glpK genes, derived from adaptation in glycerol, uses two distinct metabolic strategies to gain growth advantage. A 27-bp deletion in the rpoC gene first increases metabolic efficiency. Then, a point mutation in the glpK gene promotes growth by improving glycerol utilization but results in increased carbon wasting as overflow metabolism. In a strain carrying both mutations, these contrasting carbon/energy saving and wasting mechanisms work together to give an 89% increase in growth rate. This study provides insight into metabolic reprogramming during adaptive laboratory evolution for fast cellular growth.
    Matched MeSH terms: Escherichia coli Proteins/genetics*
  11. Rahman RN, Leow TC, Basri M, Salleh AB
    Protein Expr. Purif., 2005 Apr;40(2):411-6.
    PMID: 15766884
    The extracellular production of T1 lipase was performed by co-expression of pJL3 vector encoding bacteriocin release protein in prokaryotic system. Secretory expression was optimized by considering several parameters, including host strains, inducer (IPTG) concentration, media, induction at A(600 nm), temperature, and time of induction. Among the host strains tested, Origami B excreted out 18,100 U/ml of lipase activity into culture medium when induced with 50 microM IPTG for 12 h. The Origami B harboring recombinant plasmid pGEX/T1S and pJL3 vector was chosen for further study. IPTG at 0.05 mM, YT medium, induction at A(600 nm) of 1.25, 30 degrees C, and 32 h of induction time were best condition for T1 lipase secretion with Origami B as a host.
    Matched MeSH terms: Escherichia coli Proteins/genetics*
  12. Lim BN, Chin CF, Choong YS, Ismail A, Lim TS
    Toxicon, 2016 Jul;117:94-101.
    PMID: 27090555 DOI: 10.1016/j.toxicon.2016.04.032
    Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.
    Matched MeSH terms: Escherichia coli Proteins/immunology*
  13. Zainol MKM, Linforth RJC, Winzor DJ, Scott DJ
    Eur Biophys J, 2021 Dec;50(8):1103-1110.
    PMID: 34611772 DOI: 10.1007/s00249-021-01572-y
    This investigation of the temperature dependence of DppA interactions with a subset of three dipeptides (AA. AF and FA) by isothermal titration calorimetry has revealed the negative heat capacity ([Formula: see text]) that is a characteristic of hydrophobic interactions. The observation of enthalpy-entropy compensation is interpreted in terms of the increased structuring of water molecules trapped in a hydrophobic environment, the enthalpic energy gain from which is automatically countered by the entropy decrease associated with consequent loss of water structure flexibility. Specificity for dipeptides stems from appropriate spacing of designated DppA aspartate and arginine residues for electrostatic interaction with the terminal amino and carboxyl groups of a dipeptide, after which the binding pocket closes to become completely isolated from the aqueous environment. Any differences in chemical reactivity of the dipeptide sidechains are thereby modulated by their occurrence in a hydrophobic environment where changes in the structural state of entrapped water molecules give rise to the phenomenon of enthalpy-entropy compensation. The consequent minimization of differences in the value of ΔG0 for all DppA-dipeptide interactions thus provides thermodynamic insight into the biological role of DppA as a transporter of all dipeptides across the periplasmic membrane.
    Matched MeSH terms: Escherichia coli Proteins*
  14. Kueh R, Rahman NA, Merican AF
    J Mol Model, 2003 Apr;9(2):88-98.
    PMID: 12707802
    The arginine repressor (ArgR) of Escherichia coli binds to six L-arginine molecules that act as its co-repressor in order to bind to DNA. The binding of L-arginine molecules as well as its structural analogues is compared by means of computational docking. A grid-based energy evaluation method combined with a Monte Carlo simulated annealing process was used in the automated docking. For all ligands, the docking procedure proposed more than one binding site in the C-terminal domain of ArgR (ArgRc). Interaction patterns of ArgRc with L-arginine were also observed for L-canavanine and L-citrulline. L-lysine and L-homoarginine, on the other hand, were shown to bind poorly at the binding site. Figure A general overview of the sites found from docking the various ligands into ArgRc ( grey ribbons). Red coloured sticks: residues in binding site H that was selected for docking
    Matched MeSH terms: Escherichia coli Proteins/chemistry*
  15. Ali SA, Chew YW, Omar TC, Azman N
    PLoS One, 2015;10(12):e0144189.
    PMID: 26642325 DOI: 10.1371/journal.pone.0144189
    Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.
    Matched MeSH terms: Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism*
  16. Zaman K, Rahim F, Taha M, Wadood A, Shah SAA, Ahmed QU, et al.
    Sci Rep, 2019 11 05;9(1):16015.
    PMID: 31690793 DOI: 10.1038/s41598-019-52100-0
    Here in this study regarding the over expression of TP, which causes some physical, mental and socio problems like psoriasis, chronic inflammatory disease, tumor angiogenesis and rheumatoid arthritis etc. By this consideration, the inhibition of this enzyme is vital to secure life from serious threats. In connection with this, we have synthesized twenty derivatives of isoquinoline bearing oxadiazole (1-20), characterized through different spectroscopic techniques such as HREI-MS, 1H- NMR and 13C-NMR and evaluated for thymidine phosphorylase inhibition. All analogues showed outstanding inhibitory potential ranging in between 1.10 ± 0.05 to 54.60 ± 1.50 µM. 7-Deazaxanthine (IC50 = 38.68 ± 1.12 µM) was used as a positive control. Through limited structure activity relationships study, it has been observed that the difference in inhibitory activities of screened analogs are mainly affected by different substitutions on phenyl ring. The effective binding interactions of the most active analogs were confirmed through docking study.
    Matched MeSH terms: Escherichia coli Proteins/metabolism; Escherichia coli Proteins/chemistry
  17. Ahmad KA, Mohanmmed AS, Abas F, Chin SC
    Virol Sin, 2015 Feb;30(1):73-5.
    PMID: 25662886 DOI: 10.1007/s12250-014-3541-8
    Matched MeSH terms: Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism
  18. Nhu NTK, Phan MD, Peters KM, Lo AW, Forde BM, Min Chong T, et al.
    mBio, 2018 08 21;9(4).
    PMID: 30131362 DOI: 10.1128/mBio.01462-18
    Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis.IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infection, a disease increasingly associated with escalating antibiotic resistance. UPEC strains possess multiple surface-associated factors that enable their colonization of the urinary tract, including fimbriae, curli, and autotransporters. Curli are extracellular amyloid fibers that enhance UPEC virulence and promote biofilm formation. Here we examined the function and regulation of curli in a UPEC pyelonephritis strain belonging to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. Curli expression at human physiological temperature led to increased biofilm formation, resistance of sessile cells to the human cationic peptide LL-37, and enhanced bladder colonization. Using a comprehensive genetic screen, we identified multiple genes involved in curli production, including several that were novel or poorly characterized with respect to curli synthesis. In total, this study demonstrates an important role for curli as a UPEC virulence factor that promotes biofilm formation, resistance, and pathogenesis.
    Matched MeSH terms: Escherichia coli Proteins/genetics*; Escherichia coli Proteins/metabolism
  19. Ngaini Z, Mortadza NA
    Nat Prod Res, 2019 Dec;33(24):3507-3514.
    PMID: 29911437 DOI: 10.1080/14786419.2018.1486310
    Chemical modification of medicines from natural product-based molecules has become of interest in recent years. In this study, a series of halogenated azo derivatives 1a-d were synthesised via coupling reaction, followed by Steglich esterification with aspirin (a natural product derivative) to form azo derivatives 2a-d. While, halogenated azo-aspirin 3a-d were synthesised via direct coupling reaction of aspirin and diazonium salt. Bacteriostatic activity was demonstrated against E. coli and S. aureus via turbidimetric kinetic method. Compound 3a-d showed excellent antibacterial activities against E. coli (MIC 75-94 ppm) and S. aureus (MIC 64-89 ppm) compared to ampicillin (MIC 93 and 124 ppm respectively), followed by 1a-d and 2a-d. The presence of reactive groups of -OH, N=N, C=O and halogens significantly contribute excellent interaction towards E. coli and S. aureus. Molecular dockings analysis of 3a against MIaC protein showed binding free energy of -7.2 kcal/mol (E. coli) and -6.6 kcal/mol (S. aureus).
    Matched MeSH terms: Escherichia coli Proteins/metabolism; Escherichia coli Proteins/chemistry
  20. Goulter RM, Taran E, Gentle IR, Gobius KS, Dykes GA
    Colloids Surf B Biointerfaces, 2014 Jul 1;119:90-8.
    PMID: 24880987 DOI: 10.1016/j.colsurfb.2014.04.003
    The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.
    Matched MeSH terms: Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Escherichia coli Proteins/chemistry*
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