Displaying publications 1 - 20 of 219 in total

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  1. Fulltext Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone
    Forde BM, Phan MD, Gawthorne JA, Ashcroft MM, Stanton-Cook M, Sarkar S, et al.
    mBio, 2015 Nov 17;6(6):e01602-15.
    PMID: 26578678 DOI: 10.1128/mBio.01602-15
    Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.

    IMPORTANCE: DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.

    Matched MeSH terms: Uropathogenic Escherichia coli/genetics
  2. Fulltext Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage
    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: Uropathogenic Escherichia coli/genetics*
  3. Fulltext Novel Plasmid-Mediated Colistin Resistance Gene mcr-3 in Escherichia coli
    Yin W, Li H, Shen Y, Liu Z, Wang S, Shen Z, et al.
    mBio, 2017 06 27;8(3).
    PMID: 28655818 DOI: 10.1128/mBio.00543-17
    The mobile colistin resistance gene mcr-1 has attracted global attention, as it heralds the breach of polymyxins, one of the last-resort antibiotics for the treatment of severe clinical infections caused by multidrug-resistant Gram-negative bacteria. To date, six slightly different variants of mcr-1, and a second mobile colistin resistance gene, mcr-2, have been reported or annotated in the GenBank database. Here, we characterized a third mobile colistin resistance gene, mcr-3 The gene coexisted with 18 additional resistance determinants in the 261-kb IncHI2-type plasmid pWJ1 from porcine Escherichia colimcr-3 showed 45.0% and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively, while the deduced amino acid sequence of MCR-3 showed 99.8 to 100% and 75.6 to 94.8% identity to phosphoethanolamine transferases found in other Enterobacteriaceae species and in 10 Aeromonas species, respectively. pWJ1 was mobilized to an E. coli recipient by conjugation and contained a plasmid backbone similar to those of other mcr-1-carrying plasmids, such as pHNSHP45-2 from the original mcr-1-harboring E. coli strain. Moreover, a truncated transposon element, TnAs2, which was characterized only in Aeromonas salmonicida, was located upstream of mcr-3 in pWJ1. This ΔTnAs2-mcr-3 element was also identified in a shotgun genome sequence of a porcine E. coli isolate from Malaysia, a human Klebsiella pneumoniae isolate from Thailand, and a human Salmonella enterica serovar Typhimurium isolate from the United States. These results suggest the likelihood of a wide dissemination of the novel mobile colistin resistance gene mcr-3 among Enterobacteriaceae and aeromonads; the latter may act as a potential reservoir for mcr-3IMPORTANCE The emergence of the plasmid-mediated colistin resistance gene mcr-1 has attracted substantial attention worldwide. Here, we examined a colistin-resistant Escherichia coli isolate that was negative for both mcr-1 and mcr-2 and discovered a novel mobile colistin resistance gene, mcr-3 The amino acid sequence of MCR-3 aligned closely with phosphoethanolamine transferases from Enterobacteriaceae and Aeromonas species originating from both clinical infections and environmental samples collected in 12 countries on four continents. Due to the ubiquitous profile of aeromonads in the environment and the potential transfer of mcr-3 between Enterobacteriaceae and Aeromonas species, the wide spread of mcr-3 may be largely underestimated. As colistin has been and still is widely used in veterinary medicine and used at increasing frequencies in human medicine, the continuous monitoring of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria is imperative for understanding and tackling the dissemination of mcr genes in both the agricultural and health care sectors.
    Matched MeSH terms: Escherichia coli/genetics*
  4. Fulltext Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli
    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: Uropathogenic Escherichia coli/genetics*
  5. Fulltext T4-like coliphage ΦKAZ14 virulent to pathogenic and extended spectrum β-lactamase-producing Escherichia coli of poultry origin
    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/genetics
  6. Fulltext Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli
    Chee Wei T, Nurul Wahida AG, Shaharum S
    Trop Biomed, 2014 Dec;31(4):792-801.
    PMID: 25776606 MyJurnal
    Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.
    Matched MeSH terms: Escherichia coli/genetics
  7. Fulltext Immunogenicity of recombinant BCG-based vaccine expressing the 22 kDa of serine repeat antigen (SE22) of Plasmodium falciparum
    Teo WH, Nurul AA, Norazmi MN
    Trop Biomed, 2012 Jun;29(2):239-53.
    PMID: 22735846 MyJurnal
    The Plasmodium falciparum serine repeat antigen (SERA) is one of the promising blood-stage malarial vaccine candidates. In this study, recombinant Mycobacterium bovis bacille Calmette-Guerin (rBCG) expressing the 22 kDa protein (SE22) from the 47 kDa Nterminal domain of serine repeat antigen (SERA), generated in favour of mycobacterium codon usage, elicited specific immune response in BALB/c mice with a mixed Th1/Th2 profile. Immunized sera containing high levels of specific IgG1 and IgG2a against the epitope (as determined by ELISA) were reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA). Furthermore, the lymphocyte proliferative response to SE22 antigen from rBCG-immunized mice was higher than that of controls. The expression of intracellular cytokines (IL-2, IL-4 and IFNγ) in CD4+- and CD8+-cells was also enhanced following in-vitro stimulation with SE22. These findings indicate that a rBCG-based vaccine candidate expressing a blood-stage antigen of P. falciparum could enhance both humoral and cellular immune responses, thus paving the way for the rational use of rBCG as a vaccine candidate against malaria.
    Matched MeSH terms: Escherichia coli/genetics
  8. Fulltext Cloning, expression and protective capacity of 37 kDa outer membrane protein gene (ompH) of Pasteurella multocida serotype B:2
    Tan HY, Nagoor NH, Sekaran SD
    Trop Biomed, 2010 Dec;27(3):430-41.
    PMID: 21399583 MyJurnal
    The major outer membrane protein (OmpH) of 4 local Malaysian strains of Pasteurella multocida serotype B:2 were characterized in comparison to ATCC strains. Three major peptide bands of MW 26, 32 and 37 kDa were characterized using SDSPAGE. Two of these fragments, the 32 kDa and 37 kDa were observed to be more reactive with a mouse polyclonal antiserum in all of the local isolates as well as the ATCC strains in a Western blot. However, the 32 kDa fragment was found to cross react with other Gram negative bacteria. Therefore, the 37 kDa OmpH was selected as vaccine candidate. The 37 kDa ompH gene of the isolated strain 1710 was cloned into an Escherichia coli expression vector to produce large amounts of recombinant OmpH (rOmpH). The 37 kDa ompH gene of strain 1710 was sequenced. In comparison to a reference strain X-73 of the ompH of P. multocida, 39bp was found deleted in the 37 kDa ompH gene. However, the deletion did not shift the reading frame or change the amino acid sequence. The rOmpH was used in a mice protection study. Mice immunized and challenged intraperitoneally resulted 100% protection against P. multocida whilst mice immunized subcutaneously and challenged intraperitoneally only resulted 80% protection. The rOmpH is therefore a suitable candidate for vaccination field studies. The same rOmpH was also used to develop a potential diagnostic kit in an ELISA format.
    Matched MeSH terms: Escherichia coli/genetics
  9. Fulltext Loop-mediated isothermal amplification assay for detection of generic and verocytotoxin-producing Escherichia coli among indigenous individuals in Malaysia
    Teh CS, Chua KH, Lim YA, Lee SC, Thong KL
    ScientificWorldJournal, 2014;2014:457839.
    PMID: 24967435 DOI: 10.1155/2014/457839
    We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 10(2) CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 10(3) CFU/mL (Tt = 31.12). We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.
    Matched MeSH terms: Escherichia coli/genetics*; Shiga-Toxigenic Escherichia coli/genetics*
  10. Fulltext Engineering the production of major catechins by Escherichia coli carrying metabolite genes of Camellia sinensis
    Umar KM, Abdulkarim SM, Radu S, Abdul Hamid A, Saari N
    ScientificWorldJournal, 2012;2012:529031.
    PMID: 22645428 DOI: 10.1100/2012/529031
    A mimicked biosynthetic pathway of catechin metabolite genes from C. sinensis, consisting of flavanone 3 hydroxylase (F3H), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LCR), was designed and arranged in two sets of constructs: (a) single promoter in front of F3H and ribosome-binding sequences both in front of DFR and LCR; (b) three different promoters with each in the front of the three genes and ribosome-binding sequences at appropriate positions. Recombinant E. coli BL (DE3) harbouring the constructs were cultivated for 65 h at 26 °C in M9 medium consisting of 40 g/L glucose, 1 mM IPTG, and 3 mM eriodictyol. Compounds produced were extracted in ethyl acetate in alkaline conditions after 1 h at room temperature and identified by HPLC. Two of the four major catechins, namely, (-)-epicatechin (0.01) and (-)-epicatechin gallate (0.36 mg/L), and two other types ((+)-catechin hydrate (0.13 mg/L) and (-)-catechin gallate (0.04 mg/L)) were successfully produced.
    Matched MeSH terms: Escherichia coli/genetics*
  11. Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1
    Ang SS, Salleh AB, Chor LT, Normi YM, Tejo BA, Rahman MBA, et al.
    Protein J, 2018 04;37(2):180-193.
    PMID: 29508210 DOI: 10.1007/s10930-018-9764-z
    The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.
    Matched MeSH terms: Escherichia coli/genetics
  12. Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization
    Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE
    Protein J, 2011 Dec;30(8):581-91.
    PMID: 22001938 DOI: 10.1007/s10930-011-9365-6
    This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.
    Matched MeSH terms: Escherichia coli/genetics
  13. A new cold-adapted, organic solvent stable lipase from mesophilic Staphylococcus epidermidis AT2
    Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB
    Protein J, 2014 Jun;33(3):296-307.
    PMID: 24777627 DOI: 10.1007/s10930-014-9560-3
    The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
    Matched MeSH terms: Escherichia coli/genetics
  14. Cold-adapted RTX lipase from antarctic Pseudomonas sp. strain AMS8: isolation, molecular modeling and heterologous expression
    Ali MS, Ganasen M, Rahman RN, Chor AL, Salleh AB, Basri M
    Protein J, 2013 Apr;32(4):317-25.
    PMID: 23645400 DOI: 10.1007/s10930-013-9488-z
    A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
    Matched MeSH terms: Escherichia coli/genetics
  15. An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein
    Tan MS, Teh YH, Ho KL, Stanslas J
    Protein J, 2020 02;39(1):54-61.
    PMID: 31620959 DOI: 10.1007/s10930-019-09872-1
    Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.
    Matched MeSH terms: Escherichia coli/genetics
  16. Antimicrobial resistance and conjugative R plasmids in Escherichia coli strains isolated from animals in Peninsular Malaysia
    Koh CL, Kok CH
    Southeast Asian J. Trop. Med. Public Health, 1984 Mar;15(1):37-43.
    PMID: 6377513
    Fifteen independent E. coli strains of avian, bovine and porcine origin in Peninsular Malaysia were tested for antibiotic resistance and conjugative R plasmids. Eight (53%) isolates were found to be antibiotic resistant. Among them, 37.5% were mono-resistant and 62.5% were resistant to three or more antibiotics, i.e., multi-resistant. All of them were resistant to Tc and sensitive to Gm and Nx. Three of the eight antibiotic resistant strains were able to transfer all or part of their resistance to an E. coli K12 recipient by conjugation. The transfer frequencies of Km, Sm and Tc resistance of the three donors varied between 4.5 X 10(-8) to 6.8 X 10(-7). Analysis of the plasmid profiles of all the three donors and their respective transconjugants after agarose gel electrophoresis provided conclusive evidence that the transferable resistance traits were plasmid-mediated.
    Matched MeSH terms: Escherichia coli/genetics
  17. Nonconjugative R plasmid-mediated antibiotic resistance in Escherichia coli isolated from animals in Peninsular Malaysia
    Koh CL, Kok CH
    Southeast Asian J. Trop. Med. Public Health, 1984 Sep;15(3):313-6.
    PMID: 6395350
    Four of the five veterinary E. coli strains, which were unable to transfer their antibiotic resistance by conjugation, were found to harbour plasmids. Evidence from transformation, agarose gel electrophoresis and curing experiments showed that in strains KE-3, KE-4 and KE-14 a nonconjugative R plasmid carried the gene for resistance to tetracycline. The plasmids in KE-9 were cryptic.
    Matched MeSH terms: Escherichia coli/genetics
  18. Conjugal transfer of multiple antibiotic resistance from hospital isolates of Klebsiella to Escherichia coli
    Koh CL, Lim ME, Wong YH
    Southeast Asian J. Trop. Med. Public Health, 1983 Sep;14(3):336-40.
    PMID: 6362015
    Six independent isolates of Klebsiella from hospital environmental sources in Malaysia were found to be resistant to at least ampicillin, carbenicillin, cefoperazone, chloramphenicol, gentamicin and tetracycline. On the basis of their antibiograms, they were divided into four antibiogroups. They transferred all or part of their multiple antibiotic resistance traits to E. coli by conjugation. The results suggest that these Klebsiella strains harbour self-transmissible R plasmids. The significance of these findings are discussed.
    Matched MeSH terms: Escherichia coli/genetics*
  19. Antibiotic resistance, plasmid profile and RAPD-PCR analysis of enteropathogenic Escherichia coli (EPEC) clinical isolates
    Wan KF, Radu S, Cheah YK, Benjamin PG, Ling CM, Hon SF, et al.
    Southeast Asian J. Trop. Med. Public Health, 2003 Sep;34(3):620-6.
    PMID: 15115139
    Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea among infants in developing countries. A total of 38 EPEC isolates, obtained from diarrhea patients of Hospital Miri, Sarawak, were investigated through plasmid profile, antibiotic resistance and randomly amplified polymorphic DNA (RAPD) analysis. From the 8 types of antibiotics used, all isolates were 100% resistant to furoxime, cephalothin and sulphamethoxazole and showed high multiple antibiotic resistant (MAR) indexes, ranging from 0.5 to 1.0. In plasmid profiling, 22 isolates (58%) showed the presence of one or more plasmids in the range 1.0 to 30.9 mDa. The dendrogram obtained from the results of the RAPD-PCR discriminated the isolates into 30 single isolates and 3 clusters at the level of 40% similarity. The EPEC isolates were highly diverse, as shown by their differing plasmid profiles, antibiotic resistance patterns and RAPD profiles.
    Matched MeSH terms: Escherichia coli/genetics
  20. Mutagenicity of CORAGRAF and REKAGRAF in the Ames test
    Suzina AH, Azlina A, Shamsuria O, Samsudin AR
    Med J Malaysia, 2004 May;59 Suppl B:105-6.
    PMID: 15468840
    Mutagenicity of CORAGRAF (natural coral) and REKAGRAF (hydroxyapatite) was tested in Ames test with and without an external metabolic activation system (S9). The test revealed no mutagenic activity of both locally produced osseous substitutes.
    Matched MeSH terms: Escherichia coli/genetics
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