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Sample records for escherichia coli bacillus

  1. Enantioselective bioconversion using Escherichia coli cells expressing Saccharomyces cerevisiae reductase and Bacillus subtilis glucose dehydrogenase.

    PubMed

    Park, Hyun Joo; Jung, Jihye; Choi, Hyejeong; Uhm, Ki-Nam; Kim, Hyung Kwoun

    2010-09-01

    Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.

  2. [Factor of salinity and adaptive capacity of recombinant strains of Escherichia coli and Bacillus subtilis].

    PubMed

    Boiandin, A N; Lobova, T I; Krylova, T Iu; Kargatova, T V; Popova, L Iu; Pechurkin, N S

    2000-01-01

    Effect of different concentrations of salts on natural and recombinant strains of Bacillus subtilis and Escherichia coli was studied. The recombinant strain of B. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains of E. coli. Some salts exerted a bacteriostatic effect on E. coli and B. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.

  3. Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity

    NASA Technical Reports Server (NTRS)

    Kacena, M. A.; Smith, E. E.; Todd, P.

    1999-01-01

    The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

  4. Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var Sotto in Escherichia coli.

    PubMed

    Rady, M H

    1991-01-01

    During sporulation, Bacillus thuringiensis var. Sotto produces a parasporal crystalline protein which is toxic for the silk-worm, Bombyx mori and the cotton leaf worm, Spodoptera littoralis. The gene coding this crystal protein is present in a single plasmid. The plasmid DNA was isolated, purified and physically mapped using restriction endonuclease enzymes (R.E.). The gene coding the delta-endotoxin was inserted into Escherichia coli-Jm103, using cloning vector pUC8. Transformed E. coli cells were found to synthesize the delta-endotoxin as demonstrated by the pathogenicity of the transformed cells against 4th instar larvae of S. littoralis.

  5. Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis.

    PubMed Central

    Ohashi, H; Katsuta, Y; Nagashima, M; Kamei, T; Yano, M

    1989-01-01

    The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene. Images PMID:2504107

  6. Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli.

    PubMed

    Cho, Eun Kyung; Choi, In Soon; Choi, Young Ju

    2011-03-01

    The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino- terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. H(6)SCChi-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were 50°C and pH 8.0, respectively.

  7. Thymine auxotrophy is associated with increased UV sensitivity in Escherichia coli and Bacillus subtilis.

    PubMed

    Lojo, M M

    1995-06-01

    Thymine auxotrophy was shown to be associated with an increase in UV sensitivity both in Bacillus subtilis and in Escherichia coli. This UV sensitization became clearly evident in polA5 mutants of Bacillus subtilis: at UV doses of 16 J/m2, a reduction of more than 10-fold in the survivor population is observed in thymine requiring spontaneous mutants (polA5 thyA thyB) compared to the parental strains (polA5). Reversion of either thyA or thyB mutation led to a partial recovery in the UV resistance. This result suggests that DNA repair polymerization might be improved by the biosynthesis of thymidylate or some effect associated with such activity.

  8. Sonodynamic action of curcumin on foodborne bacteria Bacillus cereus and Escherichia coli.

    PubMed

    Wang, Xinna; Ip, Margaret; Leung, Albert Wingnang; Yang, Zhengrong; Wang, Pan; Zhang, Baoting; Ip, Siupo; Xu, Chuanshan

    2015-09-01

    Bacterial contamination is an important cause of foodborne diseases. The present study aimed to investigate sonodynamic action of curcumin on foodborne bacteria Bacillus cereus (B. cereus) and Escherichia coli (E. coli). The uptake of curcumin was measured for optimizing the concentration incubation time before ultrasound sonication, and colony forming units (CFU) were counted after ultrasound treatment. The chromosomal DNA fragmentation of bacteria was analyzed and the effect of hypoxic condition on the antibacterial efficacy of sonodynamic action of curcumin was also assessed in this study. The results showed that the maximum uptake of curcumin in B. cereus and E. coli occurred in 50min after curcumin incubation. Curcumin had sonodynamic bactericidal activity in a curcumin dose-dependent manner, and 5.6-log reduction in CFU of B. cereus was observed after curcumin treatment (2.0μM), however, only 2-log reduction in CFU of E. coli after 40μM curcumin treatment. No significant change in chromosomal DNA was found after the combined treatment of curcumin and ultrasound. The survival of B. cereus and E. coli after sonodynamic treatment in hypoxic group was significantly higher than that in normal oxygen group. These findings indicated that sonodynamic action of curcumin had significant inactivation effect on foodborne bacteria, and B. cereus was more sensitive to sonodynamic treatment of curcumin than E. coli. Sonodynamic antibacterial activity of curcumin might be dependent on the oxygen environment.

  9. Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis

    PubMed Central

    2013-01-01

    Background The anchoring motif is one of the most important aspects of cell surface display as well as efficient and stable display of target proteins. Thus, there is currently a need for the identification and isolation of novel anchoring motifs. Results A system for the display of recombinant proteins on the surface of Escherichia coli was developed using the Bacillus anthracis exosporal protein (BclA) as a new anchoring motif. For the surface display of recombinant proteins, the BAN display platform was constructed in which a target protein is linked to the C-terminus of N-terminal domain (21 amino acids) of BclA. The potential application of BAN platform for cell surface display was demonstrated with two model proteins of different size, the Bacillus sp. endoxylanase (XynA) and monooxygenase (P450 BM3m2). Through experimental analysis including outer membrane fractionation, confocal microscopy and activity assay, it was clearly confirmed that both model proteins were successfully displayed with high activities on the E. coli cell surface. Conclusions These results of this study suggest that the strategy employing the B. anthracis BclA as an anchoring motif is suitable for the display of heterologous proteins on the surface of E. coli and consequently for various biocatalytic applications as well as protein engineering. PMID:24053632

  10. Antibacterial Effects of Cissus welwitschii and Triumfetta welwitschii Extracts against Escherichia coli and Bacillus cereus

    PubMed Central

    2015-01-01

    Antibiotic resistance has increased sharply, while the pace for the development of new antimicrobials has slowed down. Plants provide an alternative source for new drugs. This study aimed to screen extracts from Cissus welwitschii and Triumfetta welwitschii for antibacterial activity against Escherichia coli and Bacillus cereus. The tests conducted included a susceptibility determination test, analysis of the effect of T. welwitschii on cell wall integrity, and transport across the membrane. It was found that the T. welwitschii methanol extracts were more effective than the water extracts and had the lowest minimum inhibitory concentration and minimum bactericidal concentration at 0.125 mg/mL and 0.5 mg/mL, respectively, against E. coli and B. cereus. The C. welwitschii extract caused the most drug accumulation in E. coli. In B. cereus, no significant drug accumulation was observed. Nucleic acid leakage in B. cereus and E. coli and protein leakage in E. coli were observed after exposure to the T. welwitschii extract. The extracts from T. welwitschii had greater antibacterial activity than the extracts from C. welwitschii. T. welwitschii may be a potential source of lead compounds for that could be developed into antibacterial agents. PMID:26904744

  11. Design and construction of a synthetic Bacillus thuringiensis Cry4Aa gene: hyperexpression in Escherichia coli.

    PubMed

    Hayakawa, Tohru; Howlader, Mohammad Tofazzal Hossain; Yamagiwa, Masashi; Sakai, Hiroshi

    2008-10-01

    Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes. However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches.

  12. Effects of Aronia melanocarpa constituents on biofilm formation of Escherichia coli and Bacillus cereus.

    PubMed

    Bräunlich, Marie; Økstad, Ole A; Slimestad, Rune; Wangensteen, Helle; Malterud, Karl E; Barsett, Hilde

    2013-12-05

    Many bacteria growing on surfaces form biofilms. Adaptive and genetic changes of the microorganisms in this structure make them resistant to antimicrobial agents. Biofilm-forming organisms on medical devices can pose serious threats to human health. Thus, there is a need for novel prevention and treatment strategies. This study aimed to evaluate the ability of Aronia melanocarpa extracts, subfractions and compounds to prevent biofilm formation and to inhibit bacterial growth of Escherichia coli and Bacillus cereus in vitro. It was found that several aronia substances possessed anti-biofilm activity, however, they were not toxic to the species screened. This non-toxic inhibition may confer a lower potential for resistance development compared to conventional antimicrobials.

  13. Analysis of insecticidal proteins from Bacillus thuringiensis and recombinant Escherichia coli by capillary electrokinetic chromatography.

    PubMed

    Luong, John H T; Male, Keith B; Mazza, Alberto; Masson, Luke; Brousseau, Roland

    2004-10-01

    Bacillus thuringiensis and recombinant Escherichia coli proteinaceous protoxins were subject to proteolysis and analyzed by capillary electrokinetic chromatography. Three resulting toxins (65 kDa) were baseline-resolved within 22 min using a 10 mM borate, pH 11 separation buffer consisting of 25 mM sodium dodecyl sulfate (SDS) and 30 mM phytic acid. The toxins displayed differential interactions with the SDS and phytic acid phases to effect their separation. The ion-pairing interaction between the analyte and phytic acid was also useful in preventing adsorption to the capillary walls and thus enhanced separation resolution and efficiency. The use of electrokinetic chromatography allows achievement of the separation in a significantly shorter time than conventional high-performance liquid chromatography (HPLC) using a diethylaminoethyl (DEAE) weak-anion exchanger.

  14. Functional and Structural Characterization of Four Glutaminases from Escherichia coli and Bacillus subtilis†

    PubMed Central

    Brown, Greg; Singer, Alex; Proudfoot, Michael; Skarina, Tatiana; Kim, Youngchang; Chang, Changsoo; Dementieva, Irina; Kuznetsova, Ekaterina; Gonzalez, Claudio F.; Joachimiak, Andrzej; Savchenko, Alexei; Yakunin, Alexander F.

    2008-01-01

    Glutaminases belong to the large superfamily of serine-dependent β-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of l-glutamine to l-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to l-glutamine and did not hydrolyze d-glutamine or l-asparagine. In each organism, one glutaminase showed higher affinity to glutamine (E. coli YbaS and B. subtilis YlaM; Km 7.3 and 7.6 mM, respectively) than the second glutaminase (E. coli YneH and B. subtilis YbgJ; Km 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical β-lactamase-like fold and conservation of several key catalytic residues of β-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme–glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli. PMID:18459799

  15. Cloning of a fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli.

    PubMed

    Ghasemi, Younes; Dabbagh, Fatemeh; Ghasemian, Abdollah

    2012-09-01

    Several investigations are being pursued to enhance the efficacy and specificity of fibrinolytic therapy. In this regard, microbial fibrinolytic enzymes attracted much more medical interests during these decades. Subtilisin, a member of subtilases (the superfamily of subtilisin-like serine proteases) and also a fibrinolytic enzyme is quite common in Gram-positive bacteria, and Bacillus species stand out in particular, as many extracellular and even intracellular variants have been identified. In the present work, the subtilisin gene from Bacillus subtilis PTCC 1023 was cloned into the vector pET-15b and expressed in Escherichia coli strain BL21 (DE3). Total genomic DNA were isolated and used for PCR amplification of the subtilisin gene by means of the specific primers. SDS-PAGE and enzyme assay were done for characterizing the expressed protein. A ~1,100 bp of the structural subtilisin gene was amplified. The DNA and amino acid sequence alignments resulting from the BLAST search of subtilisin showed high sequence identity with the other strains of B. subtilis, whereas significantly lower identity was observed with other bacterial subtilisins. The recombinant enzyme had the same molecular weight as other reported subtilisins and the E. coli transformants showed high subtilisin activity. This study provides evidence that subtilisin can be actively expressed in E. coli. The commercial availability of subtilisin is of great importance for industrial applications and also pharmaceutical purposes as thrombolytic agent. Thus, the characterization of new recombinant subtilisin and the development of rapid, simple, and effective production methods are not only of academic interest, but also of practical importance.

  16. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  17. Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment.

    PubMed

    Sherchan, Samendra P; Snyder, Shane A; Gerba, Charles P; Pepper, Ian L

    2014-01-01

    Various studies have shown that advanced oxidation processes (AOPs) such as UV light in combination with hydrogen peroxide is an efficient process for the removal of a large variety of emerging contaminants including microorganisms. The mechanism of destruction in the presence of hydrogen peroxide (H2O2) is the enhanced formation of hydroxyl (·OH) radicals, which have a high oxidation potential. The goal of this study was to utilize in-line advanced oxidation to inactivate microbes, and document the inactivation via an in-line, real-time sensor. Escherichia coli cells and Bacillus thuringiensis spores were exposed to UV/H2O2 treatment in DI water, and the online sensor BioSentry(®) was evaluated for its potential to monitor inactivation in real-time. B. thuringiensis was selected as a non-pathogenic surrogate for B. anthracis, the causative agent of anthrax and a proven biological weapon. UV radiation and UV/H2O2 exposure resulted in a >6 log10 reduction of the viable culturable counts of E. coli vegetative cells, and a 3 log10 reduction of B. thuringiensis spores. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following AOP exposure, the BioSentry sensor showed an increase in the categories of unknown, rod and spores counts, but overall, did not correspond well with viable count assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B. thuringiensis spores, which were more resistant to AOP. Further, the BioSentry in-line sensor was not successful in documenting destruction of the microbial cells in real-time.

  18. Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli

    PubMed Central

    Jameson, Katie H.; Wilkinson, Anthony J.

    2017-01-01

    Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis. PMID:28075389

  19. Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

    PubMed Central

    Juhas, Mario; Wong, Christine; Ajioka, James W.

    2016-01-01

    The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis. PMID:27798678

  20. In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases.

    PubMed Central

    Sogo, J M; Lozano, M; Salas, M

    1984-01-01

    The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase. The transcription of phage phi 29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs. Images PMID:6322128

  1. Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    McLean, K M; Whiteley, H R

    1987-01-01

    A ca. 10-kilobase (kb) HindIII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicells showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region. Images PMID:3546262

  2. Mixed culture models for predicting intestinal microbial interactions between Escherichia coli and Lactobacillus in the presence of probiotic Bacillus subtilis.

    PubMed

    Yang, J J; Niu, C C; Guo, X H

    2015-01-01

    Bacillus has been proposed as a probiotic due to its in vivo effectiveness in the gastrointestinal tract through antimicrobial activities. The present study investigates the effects of Lactobacillus alone or in the presence of Bacillus subtilis MA139 on the inhibition of pathogenic Escherichia coli K88. Mixed cultures were used to predict the possible interactions among these bacteria within the intestinal tract of animals. B. subtilis MA139 was first assayed for its inhibition against E. coli K88 both under shaking and static culture conditions. A co-culture assay was employed under static conditions to test the inhibitory effects of Lactobacillus reuteri on E. coli K88, with or without addition of B. subtilis MA139. The results showed that B. subtilis MA139 had marked inhibition against E. coli K88 under shaking conditions and weak inhibition under static conditions. Lactobacillus alone as well as in combination with B. subtilis MA139 spores exerted strong inhibition against E. coli K88 under static conditions. However, the inhibition by Lactobacillus in combination with B. subilis spores was much higher than that by Lactobacillus alone (P<0.01). B. subtilis MA139 significantly decreased the pH and oxidation-reduction potential values of the co-culture broth compared to that of Lactobacillus alone (P<0.05). The viability of Lactobacillus increased when co-cultured with B. subtilis MA139 because of significantly higher Lactobacillus counts and lower pH values in the broth (P<0.05). The role of Bacillus in the mixed culture models suggests that Bacillus may produce beneficial effects by increasing the viability of lactobacilli and subsequently inhibiting the growth of pathogenic E. coli. Therefore, the combination of Bacillus and Lactobacillus species as a probiotic is recommended.

  3. Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling.

    PubMed

    Marchand, Nicholas; Collins, Cynthia H

    2013-11-01

    The use of mixtures of microorganisms, or microbial consortia, has the potential to improve the productivity and efficiency of increasingly complex bioprocesses. However, the use of microbial consortia has been limited by our ability to control and coordinate the behaviors of microorganisms in synthetic communities. Synthetic biologists have previously engineered cell-cell communication systems that employ machinery from bacterial quorum-sensing (QS) networks to enable population-level control of gene expression. However, additional communication systems, such as those that enable communication between different species of bacteria, are needed to enable the use of diverse species in microbial consortia for bioprocessing. Here, we use the agr QS system from Staphylococcus aureus to generate an orthogonal synthetic communication system between Gram-negative Escherichia coli and Gram-positive Bacillus megaterium that is based on the production and recognition of autoinducing peptides (AIPs). We describe the construction and characterization of two types of B. megaterium "receiver" cells, capable of AIP-dependent gene expression in response to AIPs that differ by a single amino acid. Further, we observed interspecies communication when these receiver cells were co-cultured with AIP-producing E. coli. We show that the two AIP-based systems exhibit differences in sensitivity and specificity that may be advantageous in tuning communication-dependent networks in synthetic consortia. These peptide-based communication systems will enable the coordination of gene expression, metabolic pathways and growth between diverse microbial species, and represent a key step towards the use of microbial consortia in bioprocessing and biomanufacturing.

  4. Mosquito larvicidal activity of Escherichia coli with combinations of genes from Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    Ben-Dov, E; Boussiba, S; Zaritsky, A

    1995-01-01

    The genes cryIVA and cryIVD, encoding 134- and 72-kDa proteins, respectively, and the gene for a regulatory 20-kDa polypeptide of Bacillus thuringiensis subsp. israelensis (serovar H14) were cloned in all seven possible combinations by the Escherichia coli expression vectors pT7 and pUHE. The four combinations containing cryIVA (cryIVA alone, with cryIVD, with the 20-kDa-protein gene, and with both) displayed high levels of mosquito larvicidal activity in pUHE. The toxicity of the combination of cryIVA and cryIVD, with or without the 20-kDa-protein gene, was higher than has ever been achieved with delta-endotoxin genes in recombinant E. coli. Fifty percent lethal concentrations against third-instar Aedes aegypti larvae for these clones decreased (i.e., toxicity increased) continuously to about 3 x 10(5) cells ml-1 after 4 h of induction. Larvicidal activities, obtained after 30 min of induction, were lower for clones in pT7 and decreased for an additional 3.5 h. Induction of either cryIVD or the 20-kDa-protein gene alone resulted in no larvicidal activity in either pT7 or pUHE20. Cloned together, these genes were slightly toxic in pT7 but not in pUHE20. Five minutes of induction of this combination (cryIVD with the 20-kDa-protein gene) in pT7 yielded a maximal mortality of about 40%, which decreased rapidly and disappeared completely after 50 min. CryIVD is thus apparently degraded in E. coli and partially stabilized by the 20-kDa regulatory protein. Larvicidal activity of the combination of cryIVA and cryIVD was sevenfold higher than that of cryIVA alone, probably because of the cross-stabilization of the polypeptides or the synergism between their activities. PMID:7751296

  5. Fine Encapsulated ZnO Nanophosphors And Their Potential Antibacterial Evaluation On The Gram Negative Bacillus Escherichia coli

    NASA Astrophysics Data System (ADS)

    Dutta, Ranu K.; Sharma, Prashant K.; Pandey, Avinash C.

    2009-06-01

    The spherical nanophosphors of ZnO of mean size 10-20 nm were synthesized at room temperature by simple co-precipitation method. Their size and shape were governed by several encapsulations using compounds like biotin, citric acid and Poly vinyl alcohol (PVA). Besides playing a critical role in determining their size and shape, these biomolecules served as effective capping agents. The antibacterial behavior of the suspension of fine ZnO nanophosphors was investigated against the gram negative bacillus Escherichia coli. The growth curve showed bacteriostatic activity against Escherichia coli. The antibacterial activities of the ZnO nanophosphors can be attributed to their total surface area, as increasing surface to volume ratio of nanophosphors provides more efficient means for enhanced antibacterial activity.

  6. Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

    PubMed Central

    González-Candelas, L; Aristoy, M C; Polaina, J; Flors, A

    1989-01-01

    DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar. Images PMID:2515802

  7. Primary structure, expression in Escherichia coli, and properties of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from Bacillus megaterium.

    PubMed Central

    Robin, C; Blanche, F; Cauchois, L; Cameron, B; Couder, M; Crouzet, J

    1991-01-01

    A Bacillus megaterium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity was subcloned and sequenced. The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron, J. Bacteriol. 171:4222-4231, 1989). The B. megaterium polypeptide was overexpressed in Escherichia coli, partially purified, and shown to exhibit, like P. denitrificans SUMT, substrate inhibition at uroporphyrinogen III concentrations above 0.5 microM, suggesting a common regulation for aerobic cobalamin-producing organisms. Images PMID:1906874

  8. Differential analysis of bactericidal systems of blood serum with recombinant luminescent Escherichia coli and Bacillus subtilis strains.

    PubMed

    Deryabin, D G; Karimov, I F; Manukhov, I V; Tolmacheva, N A; Balabanov, V P

    2012-11-01

    Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux+ luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux+ primarily depended on the presence of β-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria.

  9. Structural and catalytic properties of CMP kinase from Bacillus subtilis: a comparative analysis with the homologous enzyme from Escherichia coli.

    PubMed

    Schultz, C P; Ylisastigui-Pons, L; Serina, L; Sakamoto, H; Mantsch, H H; Neuhard, J; Bârzu, O; Gilles, A M

    1997-04-01

    CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors with E. coli CMP kinase whereas GTP is a poor substrate with B. subtilis CMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7- to 10-fold higher in the E. coli enzyme; the relative Vm values with UMP and CMP are 0.1 for the B. subtilis CMP kinase and 0.01 for the E. coli enzyme. CMP increased the affinity of E. coli CMP kinase for ATP or for the fluorescent analog 3'-anthraniloyl dATP by one order of magnitude but had no effect on the B. subtilis enzyme. The differences in the catalytic properties of B. subtilis and E. coli CMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum of B. subtilis CMP kinase is dominated by a band at 1633 cm-1 (representing beta type structures), the spectrum of the E. coli enzyme is dominated by two bands at 1653 and 1642 cm-1 associated with alpha-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the beta-structures. ATP increases the denaturation temperature of B. subtilis CMP kinase by 9.3 degrees C, whereas in the case of the E. coli enzyme, binding of ATP has only a minor effect.

  10. Synthesis and processing of Escherichia coli TEM-beta-lactamase and Bacillus licheniformis alpha-amylase in E. coli: the role of signal peptidase I.

    PubMed

    van Dijl, J M; Smith, H; Bron, S; Venema, G

    1988-09-01

    A mutant of Escherichia coli, in which signal peptidase I synthesis can be regulated, was constructed. The mutant was used to study the effects of signal peptidase I limitation on the synthesis and efficiency of processing of two proteins: the periplasmic E. coli TEM-beta-lactamase and Bacillus licheniformis alpha-amylase, which also accumulates in the periplasm of E. coli. Signal peptidase I limitation resulted in reduced rates of processing of pre-beta-lactamase and in strong inhibition of synthesis of alpha-amylase. The data suggest that beta-lactamase is processed post-translationally and that an intimate relationship exists between the synthesis and processing of alpha-amylase.

  11. The differential stress response of adapted chromite mine isolates Bacillus subtilis and Escherichia coli and its impact on bioremediation potential.

    PubMed

    Samuel, Jastin; Paul, Madona Lien; Ravishankar, Harish; Mathur, Ankita; Saha, Dipti Priya; Natarajan, Chandrasekaran; Mukherjee, Amitava

    2013-11-01

    In the current study, indigenous bacterial isolates Bacillus subtilis VITSUKMW1 and Escherichia coli VITSUKMW3 from a chromite mine were adapted to 100 mg L(-1) of Cr(VI). The phase contrast and scanning electron microscopic images showed increase in the length of adapted E. coli cells and chain formation in case of adapted B. subtilis. The presence of chromium on the surface of the bacteria was confirmed by energy dispersive X-ray spectroscopy (EDX), which was also supported by the conspicuous Cr-O peaks in FTIR spectra. The transmission electron microscopic (TEM) images of adapted E. coli and B. subtilis showed the presence of intact cells with Cr accumulated inside the bacteria. The TEM-EDX confirmed the internalization of Cr(VI) in the adapted cells. The specific growth rate and Cr(VI) reduction capacity was significantly higher in adapted B. subtilis compared to that of adapted E. coli. To study the possible role of Cr(VI) toxicity affecting the Cr(VI) reduction capacity, the definite assays for the released reactive oxygen species (ROS) and ROS scavenging enzymes (SOD and GSH) were carried out. The decreased ROS production as well as SOD and GSH release observed in adapted B. subtilis compared to the adapted E. coli corroborated well with its higher specific growth rate and increased Cr(VI) reduction capacity.

  12. The effect of ribosomal protein S1 from Escherichia coli and Micrococcus luteus on protein synthesis in vitro by E. coli and Bacillus subtilis.

    PubMed

    Farwell, M A; Roberts, M W; Rabinowitz, J C

    1992-11-01

    We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine-Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG). These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E. coli and B. subtilis ribosomes. Translation of these nine constructions was determined with three types of ribosomes: E. coli containing ribosomal protein S1, E. coli depleted of S1, and B. subtilis which is naturally free of S1. E. coli ribosomes were able to translate all nine transcripts with variable efficiencies. B. subtilis and S1-depleted E. coli ribosomes were similar to each other and differed from non-depleted E. coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts. Addition of S1 from either E. coli or Micrococcus luteus, a Gram-positive bacterium, enabled S1-depleted E. coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B. subtilis ribosomes. AUG was the preferred initiation codon for all ribosome types; however, B. subtilis ribosomes showed greater tolerance for the non-AUG codons than either type of E. coli ribosome. The presence of a strong or stronger SD sequence increased the efficiency by which E. coli ribosomes could utilize non-AUG codons.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

    PubMed

    Aramori, I; Fukagawa, M; Tsumura, M; Iwami, M; Ono, H; Kojo, H; Kohsaka, M; Ueda, Y; Imanaka, H

    1991-12-01

    A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.

  14. Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

    PubMed Central

    Aramori, I; Fukagawa, M; Tsumura, M; Iwami, M; Ono, H; Kojo, H; Kohsaka, M; Ueda, Y; Imanaka, H

    1991-01-01

    A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis. Images FIG. 2 FIG. 5 FIG. 6 PMID:1744041

  15. How Changes in Anti-SD Sequences Would Affect SD Sequences in Escherichia coli and Bacillus subtilis.

    PubMed

    Abolbaghaei, Akram; Silke, Jordan R; Xia, Xuhua

    2017-03-31

    The 3' end of the small ribosomal RNAs (ssu rRNA) in bacteria is directly involved in the selection and binding of mRNA transcripts during translation initiation via well-documented interactions between a Shine-Dalgarno (SD) sequence located upstream of the initiation codon and an anti-SD (aSD) sequence at the 3' end of the ssu rRNA. Consequently, the 3' end of ssu rRNA (3'TAIL) is strongly conserved among bacterial species because a change in the region may impact the translation of many protein-coding genes. Escherichia coli and Bacillus subtilis differ in their 3' ends of ssu rRNA, being GAUCACCUCCUUA3' in E. coli and GAUCACCUCCUUUCU3' or GAUCACCUCCUUUCUA3' in B. subtilis Such differences in 3'TAIL lead to species-specific SDs (designated SDEc for E. coli and SDBs for B. subtilis) that can form strong and well-positioned SD/aSD pairing in one species but not in the other. Selection mediated by the species-specific 3'TAIL is expected to favour SDBs against SDEc in B. subtilis but favour SDEc against SDBs in E. coli Among well-positioned SDs, SDEc is used more in E. coli than in B. subtilis, and SDBs more in B. subtilis than in E. coli Highly expressed genes and genes of high translation efficiency tend to have longer SDs than lowly expressed genes and genes with low translation efficiency in both species, but more so in B. subtilis than in E. coli Both species overuse SDs matching the bolded part of 3'TAIL shown above. The 3'TAIL difference contributes to host-specificity of phages.

  16. Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli.

    PubMed

    de Oliveira, Rafael R; Nicholson, Wayne L

    2016-01-01

    To reduce dependence on petroleum, an alternative route to production of the chemical feedstock 2,3-butanediol (2,3-BD) from renewable lignocellulosic sources is desirable. In this communication, the genes encoding the pathway from pyruvate to 2,3-BD (alsS, alsD, and bdhA encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, respectively) from Bacillus subtilis were engineered into a single tricistronic operon under control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter in a shuttle plasmid capable of replication and expression in either B. subtilis or Escherichia coli. We describe the construction and performance of a shuttle plasmid carrying the IPTG-inducible synthetic operon alsSDbdhA coding for 2,3-BD pathway capable of (i) expression in two important representative model microorganisms, the gram-positive B. subtilis and the gram-negative E. coli; (ii) increasing 2,3-BD production in B. subtilis; and (iii) successfully introducing the B. subtilis 2,3-BD pathway into E. coli. The synthetic alsSDbdhA operon constructed using B. subtilis native genes not only increased the 2,3-BD production in its native host but also efficiently expressed the pathway in the heterologous organism E. coli. Construction of an efficient shuttle plasmid will allow investigation of 2,3-BD production performance in related organisms with industrial potential for production of bio-based chemicals.

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

    PubMed

    Juhas, Mario; Reuß, Daniel R; Zhu, Bingyao; Commichau, Fabian M

    2014-11-01

    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.

  18. Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli.

    PubMed

    Anbu Rajan, Lawrance; Joseph, Toms C; Thampuran, Nirmala; James, Roswin; Ashok Kumar, Kesavan; Viswanathan, Chinnusamy; Bansal, Kailash C

    2008-08-01

    The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.

  19. Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome.

    PubMed

    Itaya, M

    1995-07-22

    A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined. An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In the B. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in the B. subtilis chromosome and possible applications of this technique are discussed.

  20. Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli.

    PubMed

    Kim, S I; Kim, H Y; Kwak, J H; Kwon, S H; Lee, S Y

    2000-02-29

    To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.

  1. A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli.

    PubMed

    Tran, Thi Thuy; Mamo, Gashaw; Mattiasson, Bo; Hatti-Kaul, Rajni

    2010-03-01

    Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100 degrees C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20-30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed-batch cultivation, glucose concentration was maintained at 2-3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.

  2. High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

    PubMed

    Nie, Yao; Yan, Wei; Xu, Yan; Chen, Wen Bo; Mu, Xiao Qing; Wang, Xinye; Xiao, Rong

    2013-01-01

    Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful

  3. Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

    PubMed

    Nagahari, K; Sakaguchi, K

    1978-01-17

    The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu+ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B.leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu+. However, B. subtilis ilvB and ilvc auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. beta-Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.

  4. The preventive effect of Bacillus subtilus strain DB9011 against experimental infection with enterotoxcemic Escherichia coli in weaning piglets.

    PubMed

    Tsukahara, Takamitsu; Tsuruta, Takeshi; Nakanishi, Nobuo; Hikita, Chie; Mochizuki, Masami; Nakayama, Keizo

    2013-04-01

    Porcine edema disease (ED) is caused by Shiga toxin 2e-producing Escherichia coli (STEC). Post-weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial-resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilis DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21-day-old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.

  5. Experimental Research on the Sterilization of Escherichia Coli and Bacillus Subtilis in Drinking Water by Dielectric Barrier Discharge

    NASA Astrophysics Data System (ADS)

    Li, Yang; Yi, Chengwu; Li, Jingjing; Yi, Rongjie; Wang, Huijuan

    2016-02-01

    The bactericidal effect on the representative type of Gram-negative Escherichia coli (E. coli) and Gram-positive Bacillus subtilis in drinking water was investigated in this paper by using dielectric barrier discharge (DBD) advanced oxidation technology. The sterilizing rates under different conditions of reaction time t, input voltage V, pH value, and initial concentration of bacteria C0 were investigated to figure out the optimum sterilization conditions. Our observations and comparisons of cell morphology alteration by scanning electron microscopy and transmission electron microscopy revealed the sterilization mechanisms. The results showed that the sterilizing rate increased obviously with the extension of reaction time t and the rise of input voltage V. The optimal sterilization effect was achieved when the pH value was 7.1. As the initial concentration of bacteria rose, the sterilizing rate decreased. When the input voltage was 2.2 kV and the initial concentration of bacteria was relatively low, the sterilizing rate almost reached 100% after a certain treatment time in neutral aqueous solution. The reasons for the great damage of cell structure and the killing of bacteria are the oxidation of O3, OH and the accumulation of active species produced by DBD. The article provides a certain theoretical and experimental basis for DBD application in water pollution treatment. supported by the Science and Technology Support Project Plan and Social Development of Jiangsu Province, China (No. BE2011732), the Science and Technology Support Project Plan and Social Development of Zhenjiang, Jiangsu Province, China (No. SH2012013)

  6. Diarrheagenic Escherichia coli

    PubMed Central

    Nataro, James P.; Kaper, James B.

    1998-01-01

    Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens. PMID:9457432

  7. Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli.

    PubMed

    Liang, Xiaobo; Jia, Shifang; Sun, Yufang; Chen, Meiling; Chen, Xiuzhu; Zhong, Jin; Huan, Liandong

    2007-11-01

    Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l(-1) isopropyl-beta-D- thiogalactopyranoside (IPTG) at 20 degrees C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.

  8. PATHOGENIC ESCHERICHIA COLI

    EPA Science Inventory

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  9. Pathogenic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli, a member of the Enterobacteriaceae family, is a part of the normal flora of the intestinal tract of humans and a variety of animals. E. coli strains are classified on the basis of antigenic differences in two surface components (serotyping), the somatic antigen (O) of the lipopoly...

  10. Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli.

    PubMed

    Ye, Wei; Wang, Haiying; Ma, Yi; Luo, Xiaochun; Zhang, Weimin; Wang, Jufang; Wang, Xiaoning

    2013-10-10

    Glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL) is widely used in peptide recovery and synthesis because of its unique substrate specificity. However, the mechanism underlying its specificity is still not thoroughly understood. In this study, the roles of the prosegment and key amino acids involved in the proteolytic activity of GSE-BL were investigated. Loss of the GSE-BL prosegment severely restricted enzymatic activity toward Z-Phe-Leu-Glu-pNA. A homologous model of GSE-BL revealed that it contains the catalytic triad "His47, Asp96 and Ser 167", which was further confirmed by site-directed mutagenesis. In vitro mutagenesis further indicated that Val2, Arg89 and His190 are essential for enzymatic activity toward Z-Phe-Leu-Glu-pNA. Moreover, the catalytic efficiency of Phe57Ala GSE-BL toward Z-Phe-Leu-Glu-pNA was 50% higher than that of the native mature GSE-BL. This is the first study to fully elucidate the key amino acids for proteolytic activity of GSE-BL. Mature GSE-BL could be obtained through self-cleavage alone when Lys at -1 position was replaced by Glu, providing a new strategy for the preparation of mature GSE-BL. This study yielded some valuable insights into the substrate specificity of glutamate-specific endopeptidase, establishing a foundation for broadening the applications of GSE-BL.

  11. Escherichia coli biofilms

    PubMed Central

    Beloin, Christophe; Roux, Agnès; Ghigo, Jean-Marc

    2008-01-01

    Escherichia coli is a predominant species among facultative anaerobic bacteria of the gastrointestinal tract. Both its frequent community lifestyle and the availability of a wide array of genetic tools contributed to establish E. coli as a relevant model organism for the study of surface colonization. Several key factors, including different extracellular appendages, are implicated in E. coli surface colonization and their expression and activity are finely regulated, both in space and time, to ensure productive events leading to mature biofilm formation. This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli. PMID:18453280

  12. Bacillus subtilis homologs of MviN (MurJ), the putative Escherichia coli lipid II flippase, are not essential for growth.

    PubMed

    Fay, Allison; Dworkin, Jonathan

    2009-10-01

    Although peptidoglycan synthesis is one of the best-studied metabolic pathways in bacteria, the mechanism underlying the membrane translocation of lipid II, the undecaprenyl-disaccharide pentapeptide peptidoglycan precursor, remains mysterious. Recently, it was proposed that the essential Escherichia coli mviN gene encodes the lipid II flippase. Bacillus subtilis contains four proteins that are putatively homologous to MviN, including SpoVB, previously reported to be necessary for spore cortex peptidoglycan synthesis during sporulation. MviN complemented the sporulation defect of a DeltaspoVB mutation, and SpoVB and another of the B. subtilis homologs, YtgP, complemented the growth defect of an E. coli strain depleted for MviN. Thus, these B. subtilis proteins are likely to be MviN homologs. However, B. subtilis strains lacking these four proteins have no defects in growth, indicating that they likely do not serve as lipid II flippases in this organism.

  13. Diarrheagenic Escherichia coli.

    PubMed

    Gomes, Tânia A T; Elias, Waldir P; Scaletsky, Isabel C A; Guth, Beatriz E C; Rodrigues, Juliana F; Piazza, Roxane M F; Ferreira, Luís C S; Martinez, Marina B

    2016-12-01

    Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

  14. The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

    PubMed

    Kang, Tina Manzhu; Yuan, Jessica; Nguyen, Angelyn; Becket, Elinne; Yang, Hanjing; Miller, Jeffrey H

    2012-06-01

    The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

  15. The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli.

    PubMed Central

    Ploux, O; Marquet, A

    1992-01-01

    The 8-amino-7-oxopelargonate synthase [6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47] from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain. The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg. The purified enzyme is a monomer of 41 kDa. N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene. The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5'-phosphate-dependent enzymes. Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5'-phosphate. Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM. Images Fig. 1. PMID:1575677

  16. Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfpo and Escherichia coli entD genes.

    PubMed Central

    Grossman, T H; Tuckman, M; Ellestad, S; Osburne, M S

    1993-01-01

    In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin. We isolated two sets of B. subtilis DNA sequences that complemented the mutations of several E. coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes. One set contained DNA sequences that complemented only an entD mutation. The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations. The two sets of DNA sequences did not appear to overlap. AB. subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics. These data indicate that (i) at least five of the siderophore biosynthesis genes of B. subtilis can function in E. coli, (ii) the genetic organization of these siderophore genes in B. subtilis is similar to that in E. coli, and (iii) the B. subtilis entD homolog is required for efficient growth in low-iron medium. The nucleotide sequence of the B. subtilis DNA contained in plasmid pENTA22, a clone expressing the B. subtilis entD homolog, revealed the presence of at least two genes. One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B. subtilis and which is highly homologous to the E. coli entD gene. We present evidence that the E. coli entD and B. subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules. Images PMID:8407792

  17. Purification and characterization of an extracellular alpha-L-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli.

    PubMed

    Pei, Jianjun; Shao, Weilan

    2008-02-01

    The alpha-L-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50 degrees C in a minimal medium containing xylan. A 56-kDa protein with alpha-L-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was at pH 6.4 and 60 degrees C over a 10-min assay. The purified enzyme was stable over a pH range of 5.2-7.6 and had a 1-h half life at 70 degrees C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as substrate gave a K (m), and V (max) of 1.05 mM and 240 U per mg of protein. The NH(2)-terminal amino acid sequence of the enzyme was determined, and its gene araB was subsequently cloned, sequenced, and over-expressed in Escherichia coli. The open reading frame of araB consists of a 1,479-bp fragment encoding a protein of 472 amino acids, which belonged to family 51 of the glycoside hydrolases with an identity of 67% to the protein encoded by abfB of Bacillus subtilis 168.

  18. Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli.

    PubMed Central

    Visick, J E; Whiteley, H R

    1991-01-01

    CytA, a 27-kDa cytolytic crystal protein of Bacillus thuringiensis subsp. israelensis, is produced only at very low levels by recombinant Escherichia coli cells unless a 20-kDa B. thuringiensis subsp. israelensis protein is also present (K. M. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987; L. F. Adams, J. E. Visick, and H. R. Whiteley, J. Bacteriol. 171:521-530, 1989). However, the data reported here demonstrate that the 20-kDa protein is not required for high-level CytA production in E. coli strains carrying mutations in rpoH, groEL, or dnaK, all of which affect the proteolytic ability of the cells. The 20-kDa protein also increases the amount of CryIVD (another B. thuringiensis subsp. israelensis crystal protein) and LacZX90 (a mutant of beta-galactosidase) made by E. coli. The latter phenomenon is attributable to an increase in the half-life of LacZX90, suggesting that the 20-kDa protein may stabilize this protein. The effect of the 20-kDa protein was also examined in vitro and in a T7 RNA polymerase expression system, and the possible significance of these results for the timing of proteolysis and of 20-kDa protein activity is discussed. Finally, the ability of a single antibody to coimmunoprecipitate CytA and the 20-kDa protein from E. coli extracts provides evidence for a protein-protein interaction that may be related to the mechanism of action of the 20-kDa protein. Images PMID:1900280

  19. Evidence of the importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa protein cytolytic activity in Escherichia coli.

    PubMed

    Zghal, Raida Zribi; Trigui, Hana; Ben Ali, Mamdouh; Jaoua, Samir

    2008-02-01

    Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix alpha C corresponding to a substitution of Met(115) by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.

  20. Stereospecific synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli BL21 overexpressing YiaE from Escherichia coli K12 and glucose dehydrogenase from Bacillus subtilis.

    PubMed

    Yun, Hyungdon; Choi, Hyeon-Lok; Fadnavis, Nitin W; Kim, Byung-Gee

    2005-01-01

    The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.

  1. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    PubMed Central

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  2. Differential antimicrobial activity of silver nanoparticles to bacteria Bacillus subtilis and Escherichia coli, and toxicity to crop plant Zea mays and beneficial B. subtilis-inoculated Z. mays

    NASA Astrophysics Data System (ADS)

    Doody, Michael A.; Wang, Dengjun; Bais, Harsh P.; Jin, Yan

    2016-10-01

    As silver nanoparticles (AgNPs) have become increasingly used in commercial antimicrobial agents and industrial and military products, concerns are increasing over their broad environmental and health impacts and risks because they are finding their way to the environment. This study was designed to quantify the antimicrobial activity of citrate-coated AgNPs (c-AgNPs; transmission electron microscope size of 44.9 ± 7.2 nm) to two species of bacteria, i.e., Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and toxicity to a major crop plant Zea mays and beneficial bacteria-inoculated plant (i.e., B. subtilis-inoculated Z. mays symbiont). Our results reveal that the exposure of c-AgNPs significantly inhibited bacteria growth and altered their growth kinetics. Z. mays experienced significant sublethal effects including reduced root length and biomass, and hyper-accumulation of Ag in roots. The beneficial interactions between B. subtilis and Z. mays were weakened as well because both species suffered sublethal effects. Potential mechanisms leading to the antimicrobial activity and toxicity of c-AgNPs to the bacteria, plant, and plant-bacteria symbiont examined in this study were discussed. Taken together, our findings advance the current knowledge of AgNPs antimicrobial property or toxicity to bacteria, crop plant, and beneficial plant-bacteria symbiotic interaction, which is a critical component for NPs environmental impact and risk assessment.

  3. A Bacillus subtilis dnaG mutant harbours a mutation in a gene homologous to the dnaN gene of Escherichia coli.

    PubMed

    Ogasawara, N; Moriya, S; Mazza, G; Yoshikawa, H

    1986-01-01

    A dnaG mutation of Bacillus subtilis, dnaG5, was found to be linked closely to recF. We have reported previously that two putative dna genes, 'dnaA' and 'dnaN', highly homologous to Escherichia coli's dnaA and dnaN, respectively, were located adjacent to recF [Ogasawara et al., EMBO J., 4 (1985) 3345-3350]. Transformation by various fragments cloned from the 'dnaA'-recF region of the wild-type cell revealed that a 532-bp AluI fragment containing 5'-portion of the 'dnaN' gene could transform the dnaG5 mutation. The nucleotide (nt) sequence of the same fragment cloned from the mutant cell shows a single nt change in the ORF of 'dnaN' which in turn causes a single amino acid alteration from Gly to Arg. The 'dnaN' gene is now proven to be a dna gene, mutations in which result in instant arrest of chromosomal replication.

  4. Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli.

    PubMed

    Murakami, Shuichiro; Nishimoto, Haruka; Toyama, Yosuke; Shimamoto, Etsuko; Takenaka, Shinji; Kaulpiboon, Jarunee; Prousoontorn, Manchumas; Limpaseni, Tipaporn; Pongsawasdi, Piamsook; Aoki, Kenji

    2007-10-01

    A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.

  5. Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

    PubMed Central

    Gomes, P.A.D.P.; Bentancor, L.V.; Paccez, J.D.; Sbrogio-Almeida, M.E.; Palermo, M.S.; Ferreira, R.C.C.; Ferreira, L.C.S.

    2009-01-01

    No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains. PMID:24031368

  6. Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution

    SciTech Connect

    Graham, L.L.; Beveridge, T.J. )

    1990-04-01

    Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium.

  7. A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli.

    PubMed Central

    Adams, L F; Visick, J E; Whiteley, H R

    1989-01-01

    The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene. Images PMID:2644205

  8. Inactivation of Escherichia coli, Bacteriophage MS2, and Bacillus Spores under UV/H2O2 and UV/Peroxydisulfate Advanced Disinfection Conditions.

    PubMed

    Sun, Peizhe; Tyree, Corey; Huang, Ching-Hua

    2016-04-19

    Ultraviolet light (UV) combined with peroxy chemicals, such as H2O2 and peroxydisulfate (PDS), have been considered potentially highly effective disinfection processes. This study investigated the inactivation of Escherichia coli, bacteriophage MS2, and Bacillus subtilis spores as surrogates for pathogens under UV/H2O2 and UV/PDS conditions, with the aim to provide further understanding of UV-based advanced disinfection processes (ADPs). Results showed that one additional log of inactivation of E. coli was achieved with 0.3 mM H2O2 or PDS at 5.2 × 10(-5) Einstein·L(-1) photo fluence (at 254 nm) compared with UV irradiation alone. Addition of H2O2 and PDS greatly enhanced the inactivation rate of MS2 by around 15 folds and 3 folds, respectively, whereas the inactivation of B. subtilis spores was slightly enhanced. Reactive species responsible for the inactivation were identified to be •OH, SO4(·-), and CO3(·-) based on manipulation of solution conditions. The CT value of each reactive species was calculated with respect to each microbial surrogate, which showed that the disinfection efficacy ranked as •OH > SO4(·-) > CO3(·-) ≫ O2(·-)/HO2(·). A comprehensive dynamic model was developed and successfully predicted the inactivation of the microbial surrogates in surface water and wastewater matrices. The concepts of UV-efficiency and EE/O were employed to provide a cost-effective evaluation for UV-based ADPs. Overall, the present study suggests that it will be beneficial to upgrade UV disinfection to UV/H2O2 ADP for the inactivation of viral pathogens.

  9. Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy.

    PubMed

    Martinez, Keith A; Kitko, Ryan D; Mershon, J Patrick; Adcox, Haley E; Malek, Kotiba A; Berkmen, Melanie B; Slonczewski, Joan L

    2012-05-01

    The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.

  10. Survival of foodborne pathogenic bacteria (Bacillus cereus, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Listeria monocytogenes) and Bacillus cereus spores in fermented alcoholic beverages (beer and refined rice wine).

    PubMed

    Kim, S A; Kim, N H; Lee, S H; Hwang, I G; Rhee, M S

    2014-03-01

    Only limited information is available on the microbiological safety of fermented alcoholic beverages because it is still a common belief that such beverages do not provide a favorable environment for bacterial growth and survival. Thus, in this study, we examined the survival of major foodborne pathogens and spores in fermented alcoholic beverages. Foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus) and B. cereus spores (initial population, 3 to 4 log CFU/ml) were inoculated separately into three types of beer and refined rice wine, which were then stored at 5 and 22°C. Bacterial counts were assayed periodically for up to 28 days. Vegetative B. cereus counts decreased rapidly, whereas B. cereus spore counts remained constant (P > 0.05) for a long period of time in all beverages. Vegetative B. cereus cells formed spores in beer at 5 and 22°C, and the spores survived for long periods. Among vegetative cells, E. coli O157:H7 had the highest survival (only 1.49 to 1.56 log reduction during 28 days in beer at 5°C). Beer and refined rice wine supported microbial survival from several days to several weeks. Our results appear to contradict the common belief that pathogens cannot survive in alcoholic beverages. Long-term survival of pathogens (especially B. cereus and E. coli O157:H7) in beer and refined rice wine should be taken into consideration by the manufacturers of these beverages. This study provides basic information that should help further research into microbial survival in alcoholic beverages and increase the microbiological safety regulation of fermented alcoholic beverages.

  11. Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

    PubMed

    Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

    2014-03-01

    Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.

  12. Engineering Escherichia coli for methanol conversion.

    PubMed

    Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Potthoff, Eva; Heux, Stéphanie; Quax, Wim J; Wendisch, Volker F; Brautaset, Trygve; Portais, Jean-Charles; Vorholt, Julia A

    2015-03-01

    Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

  13. Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran.

    PubMed

    Lee, Eun-Jung; Lee, Bo-Hwa; Kim, Bo-Kyung; Lee, Jin-Woo

    2013-05-01

    A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.

  14. Exonuclease IX of Escherichia coli.

    PubMed Central

    Shafritz, K M; Sandigursky, M; Franklin, W A

    1998-01-01

    The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction. These enzymes are involved in replicative, repair and recombination functions. We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate. The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites. The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene. The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities. Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways. PMID:9592142

  15. Structure of Escherichia Coli Tryptophanase

    SciTech Connect

    Ku,S.; Yip, P.; Howell, P.

    2006-01-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the {alpha}-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the {alpha}-proton of the substrate for {beta}-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  16. Structure of Escherichia coli tryptophanase.

    PubMed

    Ku, Shao Yang; Yip, Patrick; Howell, P Lynne

    2006-07-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  17. Succinate production in Escherichia coli

    PubMed Central

    Thakker, Chandresh; Martínez, Irene; San, Ka-Yiu; Bennett, George N.

    2012-01-01

    Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for production of the four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon that is channeled to the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose as well as other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, as well as the requirement for efficient recovery of succinate and the reliability of the performance under scale-up are important in the overall process. The costs of the overall biorefinery compatible process will determine the economical commercialization of succinate and its impact in larger chemical markets. PMID:21932253

  18. Strategies for Protein Overproduction in Escherichia coli.

    ERIC Educational Resources Information Center

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  19. Escherichia coli survival in waters: Temperature dependence

    EPA Science Inventory

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  20. Generation of recombinant bacillus Calmette-Guérin and Mycobacterium smegmatis expressing BfpA and intimin as vaccine vectors against enteropathogenic Escherichia coli.

    PubMed

    Vasconcellos, Halyka Luzorio Franzotti; Scaramuzzi, Karina; Nascimento, Ivan Pereira; Da Costa Ferreira, Jorge M; Abe, Cecilia M; Piazza, Roxane M F; Kipnis, Andre; Dias da Silva, Wilmar

    2012-09-07

    Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in children. EPEC adheres to the intestinal epithelium and causes attaching and effacing (A/E) lesions. Recombinant Mycobacterium smegmatis (Smeg) and Mycobacterium bovis BCG strains were constructed to express either BfpA or intimin. The entire bfpA gene and a portion of the intimin gene were amplified by PCR from EPEC genomic DNA and inserted into the pMIP12 vector at the BamHI/KpnI sites. The pMIP_bfpA and pMIP_intimin vectors were introduced separately into Smeg and BCG. Recombinant clones were selected based on kanamycin resistance and designated rSmeg_pMIP_(bfpA or intimin) and rBCG_pMIP_(bfpA or intimin). The expression of bfpA and intimin was detected by Immunoblotting using polyclonal anti-BfpA and anti-intimin antibodies. The immunogenicity of these proteins was assessed in C57BL/6 mice by assaying the feces and serum for the presence of anti-BfpA and anti-intimin IgA and IgG antibodies. TNF-α and INF-γ were produced in vitro by spleen cells from mice immunized with recombinant BfpA, whereas TNF-γ was produced in mice immunized with recombinant intimin. The adhesion of EPEC (E2348/69) to HEp-2 target cells was blocked by IgA or IgG antibodies from mice immunized with recombinant BfpA or intimin but not by antibodies from non-immunized mice. Immunogenic non-infectious vectors containing relevant EPEC virulence genes may be promising vaccine candidates.

  1. Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

    PubMed

    Cammarano, P; Mazzei, F; Londei, P; Teichner, A; de Rosa, M; Gambacorta, A

    1983-08-02

    Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release r

  2. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  3. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  4. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  5. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  6. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  7. Na+/H+ antiport activity conferred by Bacillus subtilis tetA(L), a 5' truncation product of tetA(L), and related plasmid genes upon Escherichia coli.

    PubMed Central

    Cheng, J; Baldwin, K; Guffanti, A A; Krulwich, T A

    1996-01-01

    An Escherichia coli transformant expressing the Bacillus subtilis tetA(L) gene from a weak promoter was challenged by growth on medium with low, increasing tetracycline concentrations. Changes in the substrate preference ratios of the TetA(L)-mediated resistances and antiports were examined in view of recent findings suggesting that TetA(L) catalyzes efflux of Na+ in exchange for protons in addition to having the ability to catalyze metal-tetracycline/H+ antiport. After growth of the transformant on 1 microgram or more of tetracycline per ml for 12 to 15 h, the tetA(L) gene in the plasmid was found to be disrupted by an IS10 element 50 bp from the 5' end of the coding sequence. This disrupted recombinant plasmid, pKB1, conferred greater tetracycline resistance and higher levels of membrane metal-tetracycline/proton antiport than the original plasmid, pJTA1, but conferred lower NA+ resistance and Na+/H+ antiport levels than the original plasmid. The results indicate that the 5' end of the gene is necessary for optimal Na+/H+ antiport but that some such activity as well as robust tetracycline/H+ antiport persists in its absence. Two plasmid genes, tet(K) and qacA, were compared with tetA(L) vis-à-vis their abilities to enhance the Na+/H+ antiporter activity of everted vesicles from E. coli transformants. tet(K), which is more closely related to tetA(L), catalyzed 22Na+ uptake by energized vesicles, whereas the less closely related qacA gene did not. PMID:8849239

  8. Clinical Implications of Enteroadherent Escherichia coli

    PubMed Central

    Arenas-Hernández, Margarita M.P.; Martínez-Laguna, Ygnacio; Torres, Alfredo G.

    2012-01-01

    Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli enteropathogenic E. coli and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including non-intimate adherence mediated by various adhesins. These so called “enteroadherent E. coli ” categories subsequently produced toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease. PMID:22798032

  9. In-stream Escherichia coli Modeling

    NASA Astrophysics Data System (ADS)

    Pandey, P.; Soupir, M.

    2013-12-01

    Elevated levels of pathogenic bacteria indicators such as Escherichia coli (E. coli) in streams are a serious concern. Controlling E. coli levels in streams requires improving our existing understanding of fate and transport of E. coli at watershed scale. In-stream E. coli concentrations are potentially linked to non-point pollution sources (i.e., agricultural land). Water of a natural stream can receive E. coli by either through overland flow (via runoff from cropland) or resuspension from the streambed to the water column. Calculating in-stream total E. coli loads requires estimation of particle attached bacteria as well free floating E. coli transport. Currently water quality models commonly used for predicting E. coli levels in stream water have limited capability for predicting E. coli levels in the water column as well as in the streambed sediment. The challenges in calculating in-stream E. coli levels include difficulties in modeling the complex interactions between sediment particles and E. coli. Here we have developed a watershed scale model (integrated with Soil and Water Assessment Tool (SWAT)), which involves calculation of particle attached E. coli, to predict in-stream E. coli concentrations. The proposed model predicts E. coli levels in streambed bed sediment as well as in the water column. An extensive in-stream E. coli monitoring was carried out to verify the model predictions, and results indicate that the model performed well. The study proposed here will improve understanding on in-stream bacterial contamination, and help improving existing water quality models for predicting pathogenic bacteria levels in ambient water bodies.

  10. Native valve Escherichia coli endocarditis following urosepsis.

    PubMed

    Rangarajan, D; Ramakrishnan, S; Patro, K C; Devaraj, S; Krishnamurthy, V; Kothari, Y; Satyaki, N

    2013-05-01

    Gram-negative organisms are a rare cause of infective endocarditis. Escherichia coli, the most common cause of urinary tract infection and gram-negative septicemia involves endocardium rarely. In this case report, we describe infection of native mitral valve by E. coli following septicemia of urinary tract origin in a diabetic male; subsequently, he required prosthetic tissue valve replacement indicated by persistent sepsis and congestive cardiac failure.

  11. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ... Food Safety and Inspection Service Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products... manufacturing trimmings for six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45..., non-intact product, that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26,...

  12. Escherichia Coli--Key to Modern Genetics.

    ERIC Educational Resources Information Center

    Bregegere, Francois

    1982-01-01

    Mid-nineteenth century work by Mendel on plant hybrids and by Pasteur on fermentation gave birth by way of bacterial genetics to modern-day molecular biology. The bacterium Escherichia Coli has occupied a key position in genetic studies leading from early gene identification with DNA to current genetic engineering using recombinant DNA technology.…

  13. Escherichia coli and Sudden Infant Death Syndrome

    PubMed Central

    Bettelheim, Karl A.; Goldwater, Paul N.

    2015-01-01

    This review examines the association of strains of Escherichia coli with sudden infant death syndrome (SIDS) and the possible role these bacteria play in this enigmatic condition. The review addresses evidence for E. coli in SIDS infants, potential sources of E. coli in the environment, colonization by commensal and pathogenic strains, the variety of currently accepted pathotypes, and how these pathotypes could compromise intestinal integrity and induce inflammation. Both intestinal and extraintestinal pathotypes are compared in relation to the apparent liability in which virulence traits can be gained or lost by strains of E. coli. The way in which E. coli infections fit with current views on infant sleeping position and other SIDS risk factors is highlighted. PMID:26191064

  14. Synthesis and processing in Escherichia coli of human leucocyte interferon fused with the signal sequence of Bacillus amyloliquefaciens a-amylase

    SciTech Connect

    Sorokin, A.V.; Avakov, A.S.; Bogush, V.G.; Kostrov, S.V.; Gaiga, G.Z.; Iomantas, Yu.V.; Abalakina, E.G.; Stepanov, A.I.; Strongin, A.Ya.; Kozlov, Yu.I.; Debabov, V.G.

    1985-11-01

    Earlier, the authors reported cloning of the alpha-amylase gene of B. amyloliquefaciens in B. subtilis and E. coli. Currently, the authors report results on the expression of the hybrid gene consisting of the DNA fragment coding for the leader part of B. amyloliquefaciens alpha-amylase and the structural part of the human interferon alpha-2 in E. coli cells. This gene contains an additional methionine codon at the 5'-terminal, which codes for the interferon structure (without its own signal peptide). The interferon gene was inserted into plasmid /sub p/TG 278 at the cleavage site of EcoRI. The structure of the plasmid thus obtained the signal peptide of amylase, five amino acids (Val-Gly-Glu-Phe-Met), and the structural part of the interferon. The E. coli C600 cells carrying plasmid pTGA6 were used to study interferon secretions. The interferon activity was determined radioimmunologically with the use of monoclonal anti-bodies NK2.

  15. Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

    PubMed Central

    Lytle, Darren A.; Rice, Eugene W.; Johnson, Clifford H.; Fox, Kim R.

    1999-01-01

    The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased. PMID:10388724

  16. The flexible feedstock concept in Industrial Biotechnology: Metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources.

    PubMed

    Wendisch, Volker F; Brito, Luciana Fernandes; Gil Lopez, Marina; Hennig, Guido; Pfeifenschneider, Johannes; Sgobba, Elvira; Veldmann, Kareen H

    2016-09-20

    Most biotechnological processes are based on glucose that is either present in molasses or generated from starch by enzymatic hydrolysis. At the very high, million-ton scale production volumes, for instance for fermentative production of the biofuel ethanol or of commodity chemicals such as organic acids and amino acids, competing uses of carbon sources e.g. in human and animal nutrition have to be taken into account. Thus, the biotechnological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker's yeast used in these large scale processes have been engineered for efficient utilization of alternative carbon sources. This flexible feedstock concept is central to the use of non-glucose second and third generation feedstocks in the emerging bioeconomy. The metabolic engineering efforts to broaden the substrate scope of E. coli, C. glutamicum, pseudomonads, B. subtilis and yeasts to include non-native carbon sources will be reviewed. Strategies to enable simultaneous consumption of mixtures of native and non-native carbon sources present in biomass hydrolysates will be summarized and a perspective on how to further increase feedstock flexibility for the realization of biorefinery processes will be given.

  17. Hydrogen production by recombinant Escherichia coli strains

    PubMed Central

    Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

    2012-01-01

    Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995

  18. Uropathogenic Escherichia coli-Associated Exotoxins.

    PubMed

    Welch, Rodney A

    2016-06-01

    Escherichia coli are a common cause of infectious disease outside of the gastrointestinal tract. Several independently evolved E. coli clades are common causes of urinary tract and bloodstream infections. There is ample epidemiological and in vitro evidence that several different protein toxins common to many, but not all, of these strains are likely to aid the colonization and immune-evasion ability of these bacteria. This review discusses our current knowledge and areas of ignorance concerning the contribution of the hemolysin; cytotoxic-necrotizing factor-1; and the autotransporters, Sat, Pic, and Vat, to extraintestinal human disease.

  19. The evolution of the Escherichia coli phylogeny.

    PubMed

    Chaudhuri, Roy R; Henderson, Ian R

    2012-03-01

    Escherichia coli is familiar to biologists as a classical model system, ubiquitous in molecular biology laboratories around the world. Outside of the laboratory, E. coli strains exist as an almost universal component of the lower-gut flora of humans and animals. Although usually a commensal, E. coli has an alter ego as a pathogen, and is associated with diarrhoeal disease and extra-intestinal infections. The study of E. coli diversity predates the availability of molecular data, with strains initially distinguished by serotyping and metabolic profiling, and genomic diversity illustrated by DNA hybridisation. The quantitative study of E. coli diversity began with the application of multi-locus enzyme electrophoresis (MLEE), and has progressed with the accumulation of nucleotide sequence data, from single genes through multi-locus sequence typing (MLST) to whole genome sequencing. Phylogenetic methods have shed light on the processes of genomic evolution in this extraordinarily diverse species, and revealed the origins of pathogenic E. coli strains, including members of the phylogenetically indistinguishable "genus"Shigella. In May and June 2011, an outbreak of haemorrhagic uraemic syndrome in Germany was linked to a strain of enterohaemorrhagic E. coli (EHEC) O104:H4. Application of high-throughput sequencing technologies allowed the genome and origins of the outbreak strain to be characterised in real time as the outbreak was in progress.

  20. Automatic tracking of Escherichia coli bacteria.

    PubMed

    Xie, Jun; Khan, Shahid; Shah, Mubarak

    2008-01-01

    In this paper, we present an automatic method for estimating the trajectories of Escherichia coli bacteria from in vivo phase-contrast microscopy videos. To address the low-contrast boundaries in cellular images, an adaptive kernel-based technique is applied to detect cells in sequence of frames. Then a novel matching gain measure is introduced to cope with the challenges such as dramatic changes of cells' appearance and serious overlapping and occlusion. For multiple cell tracking, an optimal matching strategy is proposed to improve the handling of cell collision and broken trajectories. The results of successful tracking of Escherichia coli from various phase-contrast sequences are reported and compared with manually-determined trajectories, as well as those obtained from existing tracking methods. The stability of the algorithm with different parameter values is also analyzed and discussed.

  1. Topical Application of Escherichia coli-Vectored Vaccine as a Simple Method for Eliciting Protective Immunity

    PubMed Central

    Zhang, Jianfeng; Shi, Zhongkai; Kong, Fan-kun; Jex, Edward; Huang, Zhigang; Watt, James M.; Van Kampen, Kent R.; Tang, De-chu C.

    2006-01-01

    We report here that animals can be protected against lethal infection by Clostridium tetani cells and Bacillus anthracis spores following topical application of intact particles of live or γ-irradiated Escherichia coli vectors overproducing tetanus and anthrax antigens, respectively. Cutaneous γδT cells were rapidly recruited to the administration site. Live E. coli cells were not found in nonskin tissues after topical application, although fragments of E. coli DNA were disseminated transiently. Evidence suggested that intact E. coli particles in the outer layer of skin may be disrupted by a γδT-cell-mediated innate defense mechanism, followed by the presentation of E. coli ligand-adjuvanted intravector antigens to the immune system and rapid degradation of E. coli components. The nonreplicating E. coli vector overproducing an exogenous immunogen may foster the development of a new generation of vaccines that can be manufactured rapidly and administered noninvasively in a wide variety of disease settings. PMID:16714593

  2. Systems Metabolic Engineering of Escherichia coli.

    PubMed

    Choi, Kyeong Rok; Shin, Jae Ho; Cho, Jae Sung; Yang, Dongsoo; Lee, Sang Yup

    2017-03-01

    Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

  3. Interaction between Escherichia coli and lunar fines

    NASA Technical Reports Server (NTRS)

    Johansson, K. R.

    1983-01-01

    A sample of mature lunar fines (10084.151) was solubilized to a high degree (about 17 percent) by the chelating agent salicylic acid (0.01. M). The neutralized (pH adjusted to 7.0) leachate was found to inhibit the growth of Escherichia coli (ATCC 259922) in a minimial mineral salts glucose medium; however, the inhibition was somewhat less than that caused by neutralized salicylic acid alone. The presence of lunar fines in the minimal medium was highly stimulatory to growth of E. coli following an early inhibitory response. The bacterium survived less well in the lunar leachate than in distilled water, no doubt because of the salicylate. It was concluded that the sample of lunar soil tested has nutritional value to E. coli and that certain products of fermentation helped to solubilize the lunar soil.

  4. Production of curcuminoids in engineered Escherichia coli.

    PubMed

    Kim, Eun Ji; Cha, Mi Na; Kim, Bog-Gyu; Ahn, Joong-Hoon

    2017-03-09

    Curcumin, a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa, possesses diverse pharmacological properties including anti-inflammatory, antioxidant, antiproliferative and antiangiogenic activity. Two curcuminoids (dicinnamoylmethane and bisdemethoxycurcumin) were synthesized from glucose in Escherichia coli. PAL (phenylalanine ammonia lyase) or TAL (tyrosine ammonia lyase), along with Os4CL (p-coumaroyl-CoA ligase) and CUS (curcumin synthase), were introduced in to E. coli, and each strain produced dicinnamoylmethane or bisdemethoxycurcumin, respectively. In order to increase the production of curcuminoids in E. coli, the shikimic acid biosynthesis pathway which increases the substrates for curcuminoid biosynthesis, was engineered. Using engineered strains, the production of bisdemethoxycurcumin increased from 0.32 to 4.63 mg/L, and that of dicinnamoylmethane from 1.24 mg/L and 6.95 mg/L.

  5. Frequency-Dependent Escherichia coli Chemotaxis Behavior

    NASA Astrophysics Data System (ADS)

    Zhu, Xuejun; Si, Guangwei; Deng, Nianpei; Ouyang, Qi; Wu, Tailin; He, Zhuoran; Jiang, Lili; Luo, Chunxiong; Tu, Yuhai

    2012-03-01

    We study Escherichia coli chemotaxis behavior in environments with spatially and temporally varying attractant sources by developing a unique microfluidic system. Our measurements reveal a frequency-dependent chemotaxis behavior. At low frequency, the E. coli population oscillates in synchrony with the attractant. In contrast, in fast-changing environments, the population response becomes smaller and out of phase with the attractant waveform. These observations are inconsistent with the well-known Keller-Segel chemotaxis equation. A new continuum model is proposed to describe the population level behavior of E. coli chemotaxis based on the underlying pathway dynamics. With the inclusion of a finite adaptation time and an attractant consumption rate, our model successfully explains the microfluidic experiments at different stimulus frequencies.

  6. Thymineless death in Escherichia coli: strain specificity.

    PubMed

    Cummings, D J; Mondale, L

    1967-06-01

    Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, B(s-12), K-12 rec-21, and possibly K-12 Lon(-), all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation.

  7. Thymineless Death in Escherichia coli: Strain Specificity

    PubMed Central

    Cummings, Donald J.; Mondale, Lee

    1967-01-01

    Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, Bs−12, K-12 rec-21, and possibly K-12 Lon−, all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation. Images PMID:5337772

  8. Diversity of CRISPR loci in Escherichia coli.

    PubMed

    Díez-Villaseñor, C; Almendros, C; García-Martínez, J; Mojica, F J M

    2010-05-01

    CRISPR (clustered regularly interspaced short palindromic repeats) and CAS (CRISPR-associated sequence) proteins are constituents of a novel genetic barrier that limits horizontal gene transfer in prokaryotes by means of an uncharacterized mechanism. The fundamental discovery of small RNAs as the guides of the defence apparatus arose as a result of Escherichia coli studies. However, a survey of the system diversity in this species in order to further contribute to the understanding of the CRISPR mode of action has not yet been performed. Here we describe two CRISPR/CAS systems found in E. coli, following the analysis of 100 strains representative of the species' diversity. Our results substantiate different levels of activity between loci of both CRISPR types, as well as different target preferences and CRISPR relevances for particular groups of strains. Interestingly, the data suggest that the degeneration of one CRISPR/CAS system in E. coli ancestors could have been brought about by self-interference.

  9. Prodigiosin - A Multifaceted Escherichia coli Antimicrobial Agent

    PubMed Central

    Zorec, Maša; Stopar, David

    2016-01-01

    Despite a considerable interest in prodigiosin, the mechanism of its antibacterial activity is still poorly understood. In this work, Escherichia coli cells were treated with prodigiosin to determine its antimicrobial effect on bacterial physiology. The effect of prodigiosin was concentration dependent. In prodigiosin treated cells above MIC value no significant DNA damage or cytoplasmic membrane disintegration was observed. The outer membrane, however, becomes leaky. Cells had severely decreased respiration activity. In prodigiosin treated cells protein and RNA synthesis were inhibited, cells were elongated but could not divide. Pre-treatment with prodigiosin improved E. coli survival rate in media containing ampicillin, kanamycin and erythromycin but not phleomycin. The results suggest that prodigiosin acts as a bacteriostatic agent in E. coli cells. If prodigiosin was diluted, cells resumed growth. The results indicate that prodigiosin has distinct mode of antibacterial action in different bacteria. PMID:27612193

  10. DNA-damaging activity of patulin in Escherichia coli.

    PubMed Central

    Lee, K S; Röschenthaler, R J

    1986-01-01

    At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity. PMID:2431653

  11. A new Escherichia coli cell division gene, ftsK.

    PubMed Central

    Begg, K J; Dewar, S J; Donachie, W D

    1995-01-01

    A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer. PMID:7592387

  12. Biodegradation of Aromatic Compounds by Escherichia coli

    PubMed Central

    Díaz, Eduardo; Ferrández, Abel; Prieto, María A.; García, José L.

    2001-01-01

    Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications. PMID:11729263

  13. Biodegradation of aromatic compounds by Escherichia coli.

    PubMed

    Díaz, E; Ferrández, A; Prieto, M A; García, J L

    2001-12-01

    Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications.

  14. Oral Administration of a Select Mixture of Bacillus Probiotics Affects the Gut Microbiota and Goblet Cell Function following Escherichia coli Challenge in Newly Weaned Pigs of Genotype MUC4 That Are Supposed To Be Enterotoxigenic E. coli F4ab/ac Receptor Negative.

    PubMed

    Zhang, Wei; Zhu, Yao-Hong; Zhou, Dong; Wu, Qiong; Song, Dan; Dicksved, Johan; Wang, Jiu-Feng

    2017-02-01

    Structural disruption of the gut microbiota and impaired goblet cell function are collateral etiologic factors in enteric diseases. Low, moderate, or high doses of a Bacillus licheniformis-B. subtilis mixture (BLS mix) were orally administered to piglets of genotype MUC4 that are supposed to be F4-expressing enterotoxigenic Escherichia coli strain (F4(+) ETEC) F4ab/ac receptor negative (i.e., MUC4-resistant piglets) for 1 week before F4(+) ETEC challenge. The luminal contents were collected from the mucosa of the colon on day 8 after F4(+) ETEC challenge. The BLS mix attenuated E. coli-induced expansion of Bacteroides uniformis, Eubacterium eligens, Acetanaerobacterium, and Sporobacter populations. Clostridium and Turicibacter populations increased following F4(+) ETEC challenge in pigs pretreated with low-dose BLS mix. Lactobacillus gasseri and Lactobacillus salivarius populations increased after administration of BLS mix during E. coli infection. The beneficial effects of BLS mix were due in part to the expansion of certain Clostridium, Lactobacillus, and Turicibacter populations, with a corresponding increase in the number of goblet cells in the ileum via upregulated Atoh1 expression, in turn increasing MUC2 production and thus preserving the mucus barrier and enhancing host defenses against enteropathogenic bacteria. However, excessive BLS mix consumption may increase the risk for enteritis, partly through disruption of colonic microbial ecology, characterized by expansion of Proteobacteria and impaired goblet cell function in the ileum. Our findings suggest that oral administration of BLS mix reprograms the gut microbiota and enhances goblet cell function to ameliorate enteritis.

  15. The 503nm pigment of Escherichia coli

    PubMed Central

    Kamitakahara, Joyce R.; Polglase, W. J.

    1970-01-01

    The yield of cell protein was one-third less for streptomycin-dependent Escherichia coli B than for the wild-type parent strain when both were grown aerobically on a medium with limiting glucose, but anaerobically the yield of protein was similar for both strains. The transient pigment absorbing at 503nm that is known to be present in E. coli and other organisms was not detectable in streptomycin-dependent mutants nor in a non-dependent (energy-deficient) revertant. When wild-type E. coli B was grown on limiting glucose–salts medium containing 2,4 dinitrophenol, the yield of cell protein was decreased and formation of the 503nm pigment was inhibited. Fumarase, aconitase and glucose 6-phosphate dehydrogenase were de-repressed in E. coli B cells grown with excess of glucose in a medium containing 2,4-dinitrophenol. In air-oxidized, wild-type E. coli B cells, the 503nm pigment appeared before reduced cytochromes when gluconate was the substrate but failed to appear when succinate was the substrate. The results provide evidence for a role of the 503nm pigment in aerobic energy metabolism, possibly as an electron acceptor from NADPH. PMID:4395501

  16. ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

    EPA Science Inventory

    The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

  17. Designed phosphoprotein recognition in Escherichia coli.

    PubMed

    Sawyer, Nicholas; Gassaway, Brandon M; Haimovich, Adrian D; Isaacs, Farren J; Rinehart, Jesse; Regan, Lynne

    2014-11-21

    Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide-protein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide-protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.

  18. Escherichia coli growth under modeled reduced gravity

    NASA Technical Reports Server (NTRS)

    Baker, Paul W.; Meyer, Michelle L.; Leff, Laura G.

    2004-01-01

    Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

  19. Detection of Escherichia coli enterotoxins in stools.

    PubMed Central

    Merson, M H; Yolken, R H; Sack, R B; Froehlich, J L; Greenberg, H B; Huq, I; Black, R W

    1980-01-01

    We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method. PMID:6995331

  20. Escherichia coli O157:H7.

    PubMed

    Mead, P S; Griffin, P M

    1998-10-10

    Escherichia coli O157 was first identified as a human pathogen in 1982. One of several Shiga toxin-producing serotypes known to cause human illness, the organism probably evolved through horizontal acquisition of genes for Shiga toxins and other virulence factors. E. coli O157 is found regularly in the faeces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact with infected people or animals. Human infection is associated with a wide range of clinical illness, including asymptomatic shedding, non-bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome, and death. Since laboratory practices vary, physicians need to know whether laboratories in their area routinely test for E. coli O157 in stool specimens. Treatment with antimicrobial agents remains controversial: some studies suggest that treatment may precipitate haemolytic uraemic syndrome, and other studies suggest no effect or even a protective effect. Physicians can help to prevent E. coli O157 infections by counselling patients about the hazards of consuming undercooked ground meat or unpasteurised milk products and juices, and about the importance of handwashing to prevent the spread of diarrhoeal illness, and by informing public-health authorities when they see unusual numbers of cases of bloody diarrhoea or haemolytic uraemic syndrome.

  1. Transport proteins promoting Escherichia coli pathogenesis.

    PubMed

    Tang, Fengyi; Saier, Milton H

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies.

  2. Transport proteins promoting Escherichia coli pathogenesis

    PubMed Central

    Tang, Fengyi; Saier, Milton H.

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. PMID:24747185

  3. Extracellular recombinant protein production from Escherichia coli.

    PubMed

    Ni, Ye; Chen, Rachel

    2009-11-01

    Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

  4. Engineering Escherichia coli to bind to cyanobacteria.

    PubMed

    Zhang, Zijian; Meng, Liuyi; Ni, Congjian; Yao, Lanqiu; Zhang, Fengyu; Jin, Yuji; Mu, Xuelang; Zhu, Shiyu; Lu, Xiaoyu; Liu, Shiyu; Yu, Congyu; Wang, Chenggong; Zheng, Pu; Wu, Jie; Kang, Li; Zhang, Haoqian M; Ouyang, Qi

    2017-03-01

    We engineered Escherichia coli cells to bind to cyanobacteria by heterologously producing and displaying lectins of the target cyanobacteria on their surface. To prove the efficacy of our approach, we tested this design on Microcystis aeruginosa with microvirin (Mvn), the lectin endogenously produced by this cyanobacterium. The coding sequence of Mvn was C-terminally fused to the ice nucleation protein NC (INPNC) gene and expressed in E. coli. Results showed that E. coli cells expressing the INPNC::Mvn fusion protein were able to bind to M. aeruginosa and the average number of E. coli cells bound to each cyanobacterial cell was enhanced 8-fold. Finally, a computational model was developed to simulate the binding reaction and help reconstruct the binding parameters. To our best knowledge, this is the first report on the binding of two organisms in liquid culture mediated by the surface display of lectins and it may serve as a novel approach to mediate microbial adhesion.

  5. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

    PubMed

    Whitaker, W Brian; Jones, J Andrew; Bennett, R Kyle; Gonzalez, Jacqueline E; Vernacchio, Victoria R; Collins, Shannon M; Palmer, Michael A; Schmidt, Samuel; Antoniewicz, Maciek R; Koffas, Mattheos A; Papoutsakis, Eleftherios T

    2017-01-01

    Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using (13)C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.

  6. Humoral immune responses of amphioxus Branchiostoma belcheri to challenge with Escherichia coli.

    PubMed

    Pang, Qiuxiang; Zhang, Shicui; Liu, Xuemei; Wu, Di

    2006-08-01

    Humoral parameters of amphioxus Branchiostoma belcheri, including lysozyme, antimicrobial activity, microbial agglutinin and haemagglutinins were measured before and after challenge with Escherichia coli. Humoral fluids from unchallenged B. belcheri had lysozyme, antimicrobial, microbial agglutinating and haemagglutinating activities, which may represent part of the baseline level of innate immunity in this organism. After challenge with E. coli, the lysozyme activity, growth-inhibiting activities against E. coli and Vibrio alginolyticus, microbial agglutinating activities against Micrococcus lysodeikticus, Bacillus subtilis and Staphylococus aureus, and haemagglutinating activities against rabbit and human A and O erythrocytes in the humoral fluids were all increased significantly. In contrast, the agglutinating activities against Vibrio harveyi and E. coli and the haemagglutinating activity against human B erythrocytes in the humoral fluids were reduced in response to E. coli challenge. It appears that the humoral fluids of B. belcheri contain components that are able to differentiate different microbes and different human blood cell types.

  7. Arabidopsis alternative oxidase sustains Escherichia coli respiration.

    PubMed Central

    Kumar, A M; Söll, D

    1992-01-01

    Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. Images PMID:1438286

  8. Role of Escherichia coli in Biofuel Production

    PubMed Central

    Koppolu, Veerendra; Vasigala, Veneela KR

    2016-01-01

    Increased energy consumption coupled with depleting petroleum reserves and increased greenhouse gas emissions have renewed our interest in generating fuels from renewable energy sources via microbial fermentation. Central to this problem is the choice of microorganism that catalyzes the production of fuels at high volumetric productivity and yield from cheap and abundantly available renewable energy sources. Microorganisms that are metabolically engineered to redirect renewable carbon sources into desired fuel products are contemplated as best choices to obtain high volumetric productivity and yield. Considering the availability of vast knowledge in genomic and metabolic fronts, Escherichia coli is regarded as a primary choice for the production of biofuels. Here, we reviewed the microbial production of liquid biofuels that have the potential to be used either alone or in combination with the present-day fuels. We specifically highlighted the metabolic engineering and synthetic biology approaches used to improve the production of biofuels from E. coli over the past few years. We also discussed the challenges that still exist for the biofuel production from E. coli and their possible solutions. PMID:27441002

  9. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1989-01-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. We are also investigating the control of other genes required for fermentation and anaerobic growth. We have isolated both structural and regulatory mutations affecting the expression of alcohol dehydrogenase, the enzyme responsible for the final step in alcohol synthesis. Some of these regulatory mutations also affect other anaerobically induced genes. The adh gene has been cloned and sequenced. The ADH protein is one of the largest highly expressed proteins in E. coli and requires approximately 2700bp of DNA for its cloning sequence. We have also isolated mutations affecting the fermentative lactate dehydrogenase. In consequence it is now possible to construct E. coli strains defective in the production of any one or more of their normal fermentation products (i.e. formate, acetate, lactate, ethanol and succinate). The factors affecting the ratio of fermentation products are being investigated by in vivo NMR spectroscopy.

  10. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1990-01-01

    The purpose of this project is to elucidate the way in which the synthesis of ethanol and related fermentation products are regulated in the facultative anaerobe Escherichia coli. We are also investigating the control of other genes required for anaerobic growth. We have isolated both structural and regulatory mutations affecting the expression of alcohol dehydrogenase, the enzyme responsible for the final step in alcohol synthesis. Some of these regulatory mutations also affect other anaerobically induced genes. The adh gene has been cloned and sequenced. The ADH protein is one of the largest highly expressed proteins in E. coli and requires approximately 2700bp of DNA for its coding sequence. We have also isolated mutations affecting the fermentative lactate dehydrogenase and have recently cloned the ldh gene. In consequence it is now possible to construct E. coli strains defective in the production of any one or more of their normal fermentation products (i.e. formate, acetate, lactate, ethanol and succinate). The factors affecting ratio of fermentation products are being investigated by in vivo NMR spectroscopy.

  11. Long term effects of Escherichia coli mastitis.

    PubMed

    Blum, Shlomo E; Heller, Elimelech D; Leitner, Gabriel

    2014-07-01

    Escherichia coli is one of the most frequently diagnosed causes of bovine mastitis, and is typically associated with acute, clinical mastitis. The objective of the present study was to evaluate the long term effects of intramammary infections by E. coli on milk yield and quality, especially milk coagulation. Twenty-four Israeli Holstein cows diagnosed with clinical mastitis due to intramammary infection by E. coli were used in this study. Mean lactation number, days in milk (DIM) and daily milk yield (DMY) at the time of infection was 3.3 ± 1.3, 131.7 days ± 78.6 and 45.7 L ± 8.4, respectively. DMY, milk constituents, somatic cells count (SCC), differential leukocytes count and coagulation parameters were subsequently assessed. Two patterns of inflammation were identified: 'short inflammation', characterized by <15% decrease in DMY and <30 days until return to normal (n = 5), and 'long inflammation', characterized by >15% decrease in DMY and >30 days to reach a new maximum DMY (n = 19). The estimated mean loss of marketable milk during the study was 200 L/cow for 'short inflammation' cases, and 1,500 L/cow for 'long inflammation' ones. Significant differences between 'short' and 'long inflammation' effects were found in almost all parameters studied. Long-term detrimental effects on milk quality were found regardless of clinical or bacteriological cure of affected glands.

  12. WGS accurately predicts antimicrobial resistance in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives: To determine the effectiveness of whole-genome sequencing (WGS) in identifying resistance genotypes of multidrug-resistant Escherichia coli (E. coli) and whether these correlate with observed phenotypes. Methods: Seventy-six E. coli strains were isolated from farm cattle and measured f...

  13. Mechanism of Escherichia coli Resistance to Pyrrhocoricin

    PubMed Central

    Narayanan, Shalini; Modak, Joyanta K.; Ryan, Catherine S.; Garcia-Bustos, Jose; Davies, John K.

    2014-01-01

    Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10−7. Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials. PMID:24590485

  14. Animal models of enteroaggregative Escherichia coli infection

    PubMed Central

    Philipson, Casandra W.; Bassaganya-Riera, Josep; Hontecillas, Raquel

    2013-01-01

    Enteroaggregative Escherichia coli (EAEC) has been acknowledged as an emerging cause of gastroenteritis worldwide for over two decades. Epidemiologists are revealing the role of EAEC in diarrheal outbreaks as a more common occurrence than ever suggested before. EAEC induced diarrhea is most commonly associated with travelers, children and immunocompromised individuals however its afflictions are not limited to any particular demographic. Many attributes have been discovered and characterized surrounding the capability of EAEC to provoke a potent pro-inflammatory immune response, however cellular and molecular mechanisms underlying initiation, progression and outcomes are largely unknown. This limited understanding can be attributed to heterogeneity in strains and the lack of adequate animal models. This review aims to summarize current knowledge about EAEC etiology, pathogenesis and clinical manifestation. Additionally, current animal models and their limitations will be discussed along with the value of applying systems-wide approaches such as computational modeling to study host-EAEC interactions. PMID:23680797

  15. An overview of atypical enteropathogenic Escherichia coli.

    PubMed

    Hernandes, Rodrigo T; Elias, Waldir P; Vieira, Mônica A M; Gomes, Tânia A T

    2009-08-01

    The enteropathogenic Escherichia coli (EPEC) pathotype is currently divided into two groups, typical EPEC (tEPEC) and atypical EPEC (aEPEC). The property that distinguishes these two groups is the presence of the EPEC adherence factor plasmid, which is only found in tEPEC. aEPEC strains are emerging enteropathogens that have been detected worldwide. Herein, we review the serotypes, virulence properties, genetic relationships, epidemiology, reservoir and diagnosis of aEPEC, including those strains not belonging to the classical EPEC serogroups (nonclassical EPEC serogroups). The large variety of serotypes and genetic virulence properties of aEPEC strains from nonclassical EPEC serogroups makes it difficult to determine which strains are truly pathogenic.

  16. Escherichia coli fliAZY operon.

    PubMed Central

    Mytelka, D S; Chamberlin, M J

    1996-01-01

    We have cloned the Escherichia coli fliAZY operon, which contains the fliA gene (the alternative sigma factor sigma F) and two novel genes, fliZ and fliY. Transcriptional mapping of this operon shows two start sites, one of which is preceded by a canonical E sigma F-dependent consensus and is dependent on sigma F for expression in vivo and in vitro. We have overexpressed and purified sigma F and demonstrated that it can direct core polymerase to E sigma F-dependent promoters. FliZ and FliY are not required for motility but may regulate sigma F activity, perhaps in response to a putative cell density signal that may be detected by FliY, a member of the bacterial extracellular solute-binding protein family 3. PMID:8550423

  17. Expanding ester biosynthesis in Escherichia coli.

    PubMed

    Rodriguez, Gabriel M; Tashiro, Yohei; Atsumi, Shota

    2014-04-01

    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l(-1)). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.

  18. [Enteroinvasive Escherichia coli. Pathogenesis and epidemiology].

    PubMed

    Prats, G; Llovet, T

    1995-03-01

    Enteroinvasive Escherichia coli (EIEC) is an intestinal pathogen causing enteritis, with a similar pathogenic mechanism to that of Shigella, which causes an epithelial invasion of the large bowel leading to inflammation and ulceration of the mucosa. The patients often develop the symptoms of bacillary dysentery. The EIEC strains are atypical in their biochemical reactions and may ferment lactose late or not at all, are lysine decarboxilase negative, and non motile. In addition, most EIEC strains express somatic antigens which are either strongly related or identical to Shigella antigens. EIEC invasion is mediated by a large plasmid (140 MDa) coding for the production of several outer membrane proteins involved in invasiveness. These strains have been isolated with some regularity in South America, the Extreme Orient, and Eastern Europe. In Spain the incidence of enteroinvasive E. coli is extraordinarily low (0.2%), the serogroup O124 being the most frequently isolated. EIEC enteritis has been associated to sporadic cases occurring in travellers. Occasional outbreaks related to ingestion of contaminated water or food and person to person have been reported.

  19. Independence of replisomes in Escherichia coli chromosomalreplication

    SciTech Connect

    Breier, Adam M.; Weier, Heinz-Ulrich G.; Cozzarelli, Nicholas R.

    2005-03-13

    In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.

  20. Nucleotide excision repair in Escherichia coli.

    PubMed Central

    Van Houten, B

    1990-01-01

    One of the best-studied DNA repair pathways is nucleotide excision repair, a process consisting of DNA damage recognition, incision, excision, repair resynthesis, and DNA ligation. Escherichia coli has served as a model organism for the study of this process. Recently, many of the proteins that mediate E. coli nucleotide excision have been purified to homogeneity; this had led to a molecular description of this repair pathway. One of the key repair enzymes of this pathway is the UvrABC nuclease complex. The individual subunits of this enzyme cooperate in a complex series of partial reactions to bind to and incise the DNA near a damaged nucleotide. The UvrABC complex displays a remarkable substrate diversity. Defining the structural features of DNA lesions that provide the specificity for damage recognition by the UvrABC complex is of great importance, since it represents a unique form of protein-DNA interaction. Using a number of in vitro assays, researchers have been able to elucidate the action mechanism of the UvrABC nuclease complex. Current research is devoted to understanding how these complex events are mediated within the living cell. PMID:2181258

  1. Chemotaxis Toward Sugars in Escherichia coli

    PubMed Central

    Adler, Julius; Hazelbauer, Gerald L.; Dahl, M. M.

    1973-01-01

    Using a quantitative assay for measuring chemotaxis, we tested a variety of sugars and sugar derivatives for their ability to attract Escherichia coli bacteria. The most effective attractants, i.e., those that have thresholds near 10−5 M or below, are N-acetyl-d-glucosamine, 6-deoxy-d-glucose, d-fructose, d-fucose, 1-d-glycerol-β-d-galactoside, galactitol, d-galactose, d-glucosamine, d-glucose, α-d-glucose-1-phosphate, lactose, maltose, d-mannitol, d-mannose, methyl-β-d-galactoside, methyl-β-d-glucoside, d-ribose, d-sorbitol, and trehalose. Lactose, and probably d-glucose-1-phosphate, are attractive only after conversion to the free monosaccharide, while the other attractants do not require breakdown for taxis. Nine different chemoreceptors are involved in detecting these various attractants. They are called the N-acetyl-glucosamine, fructose, galactose, glucose, maltose, mannitol, ribose, sorbitol, and trehalose chemoreceptors; the specificity of each was studied. The chemoreceptors, with the exception of the one for d-glucose, are inducible. The galactose-binding protein serves as the recognition component of the galactose chemoreceptor. E. coli also has osmotically shockable binding activities for maltose and d-ribose, and these appear to serve as the recognition components for the corresponding chemoreceptors. PMID:4580570

  2. Expanding ester biosynthesis in Escherichia coli

    PubMed Central

    Rodriguez, Gabriel M; Tashiro, Yohei; Atsumi, Shota

    2015-01-01

    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l−1). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters. PMID:24609358

  3. The thermal impulse response of Escherichia coli

    PubMed Central

    Paster, Eli; Ryu, William S.

    2008-01-01

    Swimming Escherichia coli responds to changes in temperature by modifying its motor behavior. Previous studies using populations of cells have shown that E. coli accumulate in spatial thermal gradients, but these experiments did not cleanly separate thermal responses from chemotactic responses. Here we have isolated the thermal response by studying the behavior of single, tethered cells. The motor output of cells grown at 33°C was measured at constant temperature, from 10° to 40°C, and in response to small, impulsive increases in temperature, from 23° to 43°C. The thermal impulse response at temperatures < 31°C is similar to the chemotactic impulse response: Both follow a similar time course, share the same directionality, and show biphasic characteristics. At temperatures > 31°C, some cells show an inverted response, switching from warm- to cold-seeking behavior. The fraction of inverted responses increases nonlinearly with temperature, switching steeply at the preferred temperature of 37°C. PMID:18385380

  4. Cyclomodulins in urosepsis strains of Escherichia coli.

    PubMed

    Dubois, Damien; Delmas, Julien; Cady, Anne; Robin, Frédéric; Sivignon, Adeline; Oswald, Eric; Bonnet, Richard

    2010-06-01

    Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (P, <0.001 to 0.005). However, the prevalences of the pks island among phylogroup B2 strains, in contrast to those of the cnf1 gene, were not significantly different between fecal and urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.

  5. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  6. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

    PubMed Central

    Ihssen, Julian; Reiss, Renate; Luchsinger, Ronny; Thöny-Meyer, Linda; Richter, Michael

    2015-01-01

    Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development. PMID:26068013

  7. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.

    PubMed

    Ihssen, Julian; Reiss, Renate; Luchsinger, Ronny; Thöny-Meyer, Linda; Richter, Michael

    2015-06-12

    Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development.

  8. Escherichia coli survival in waters: temperature dependence.

    PubMed

    Blaustein, R A; Pachepsky, Y; Hill, R L; Shelton, D R; Whelan, G

    2013-02-01

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q₁₀ model. This suggestion was made 34 years ago based on 20 survival curves taken from published literature, but has not been revisited since then. The objective of this study was to re-evaluate the accuracy of the Q₁₀ equation, utilizing data accumulated since 1978. We assembled a database of 450 E. coli survival datasets from 70 peer-reviewed papers. We then focused on the 170 curves taken from experiments that were performed in the laboratory under dark conditions to exclude the effects of sunlight and other field factors that could cause additional variability in results. All datasets were tabulated dependencies "log concentration vs. time." There were three major patterns of inactivation: about half of the datasets had a section of fast log-linear inactivation followed by a section of slow log-linear inactivation; about a quarter of the datasets had a lag period followed by log-linear inactivation; and the remaining quarter were approximately linear throughout. First-order inactivation rate constants were calculated from the linear sections of all survival curves and the data grouped by water sources, including waters of agricultural origin, pristine water sources, groundwater and wells, lakes and reservoirs, rivers and streams, estuaries and seawater, and wastewater. Dependency of E. coli inactivation rates on temperature varied among the water sources. There was a significant difference in inactivation rate values at the reference temperature between rivers and agricultural waters, wastewaters and agricultural waters, rivers and lakes, and wastewater and lakes. At specific sites, the Q₁₀ equation was more accurate in rivers and coastal waters than in lakes making the value of

  9. Polymorphisms in the umuDC region of Escherichia species. [Escherichia coli; Escherichia alkalescens; Escherichia dispar; Escherichia aurescens

    SciTech Connect

    Sedgwick, S.G.; Robson, M.; Malik, F.

    1988-04-01

    The umuDC operon of Escherichia coli encodes mutagenic DNA repair. The umuDC regions of multiple isolates of E. coli, E. alkalescens, and E. dispar and a single stock of E. aurescens were mapped by nucleotide hybridization. umuDC is located at one end of a conserved tract of restriction endonuclease sites either 12.5 or 14 kilobase pairs long. Rearrangements, including possible deletions, were seen in the polymorphic DNA flanking the conserved tract. Restriction site polymorphisms were not found around the DNA repair gene recA or polA. The junctions of the conserved region contain direct repeats of nucleotide sequences resembling the termini of the Tn3 group of transposons. Possible mechanisms for the generation of these variants are discussed.

  10. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    Sewage samples from seven locations in the United States were analyzed for Escherichia coli isolates which were resistant to trimethoprim-sulfamethoxazole (SXT). The prevalence rate of SXT resistant organisms varied between the different geographical locales. The majority of th...

  11. Genome Sequence of Escherichia coli Tailed Phage Utah

    PubMed Central

    Leavitt, Justin C.; Heitkamp, Alexandra J.; Bhattacharjee, Ananda S.; Gilcrease, Eddie B.

    2017-01-01

    ABSTRACT Escherichia coli bacteriophage Utah is a member of the chi-like tailed phage cluster in the Siphoviridae family. We report here the complete 59,024-bp sequence of the genome of phage Utah. PMID:28360173

  12. Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia.

    PubMed

    Zuo, Guanghong; Xu, Zhao; Hao, Bailin

    2013-02-01

    Shigella species and Escherichia coli are closely related organisms. Early phenotyping experiments and several recent molecular studies put Shigella within the species E. coli. However, the whole-genome-based, alignment-free and parameter-free CVTree approach shows convincingly that four established Shigella species, Shigella boydii, Shigella sonnei, Shigella felxneri and Shigella dysenteriae, are distinct from E. coli strains, and form sister species to E. coli within the genus Escherichia. In view of the overall success and high resolution power of the CVTree approach, this result should be taken seriously. We hope that the present report may promote further in-depth study of the Shigella-E. coli relationship.

  13. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI.

    PubMed

    FREIFELDER, D; MAALOE, O

    1964-10-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987-990. 1964.-Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process.

  14. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI

    PubMed Central

    Freifelder, David; Maaløe, Ole

    1964-01-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987–990. 1964.—Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process. PMID:14219063

  15. Escherichia coli Unsaturated Fatty Acid Synthesis

    PubMed Central

    Feng, Youjun; Cronan, John E.

    2009-01-01

    Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA. PMID:19679654

  16. Mono and diterpene production in Escherichia coli.

    PubMed

    Reiling, K Kinkead; Yoshikuni, Yasuo; Martin, Vincent J J; Newman, Jack; Bohlmann, Jörg; Keasling, Jay D

    2004-07-20

    Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

  17. Microdiesel: Escherichia coli engineered for fuel production.

    PubMed

    Kalscheuer, Rainer; Stölting, Torsten; Steinbüchel, Alexander

    2006-09-01

    Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli. This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l(-1) and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.

  18. The Escherichia coli Peripheral Inner Membrane Proteome*

    PubMed Central

    Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

    2013-01-01

    Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with

  19. Regulation of Glutamine Transport in Escherichia coli.

    PubMed Central

    Willis, R C; Iwata, K K; Furlong, C E

    1975-01-01

    The formation of the high-affinity (Km equal to 0.2 muM) L-glutamine transport system of Escherichia coli strain 7 (Lin) appears to be subject to the same major control as the glutamine synthetase (EC 6.3.1.2) of this gram-negative organism. Culture of cells under nitrogen-limited conditions provides maximum derepression of both the glutamine synthetase and the glutamine transport system. Nutritional conditions providing a rich supply of ammonium salts or available sources of nitrogen, i.e., conditions which repress the formation of glutamine synthetase, provide three- and 20-fold repression, respectively, of the glutamine transport system. Culture of cells with glutamine supplements of 2 mM does not increase the repression of high-affinity glutamine transport system beyond the level observed in the absence of glutamine. A second kinetically distinct low-affinity component of glutamine. A second kinetically distinct low-affinity component of glutamine uptake is observed in cells cultured with a glutamine-depleted nutrient broth. This second component is associated with the appearance of glutaminase A (EC 3.5.1.2) and asparaginase I (EC 3.5.1.1), a periplasmic enzyme. Parallel changes were observed in the levels of the high-affinity glutamine transport system and the glutamine synthetase when cells were cultured with the carbon sources: glucose, glycerol, or succinate. PMID:238938

  20. ESCHERICHIA COLI Gene Induction by Alkylation Treatment

    PubMed Central

    Volkert, Michael R.; Nguyen, Dinh C.; Beard, K. Christopher

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased β-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes. PMID:3080354

  1. Escherichia coli gene induction by alkylation treatment.

    PubMed

    Volkert, M R; Nguyen, D C; Beard, K C

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased beta-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes.

  2. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1986-03-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. Focus is on the two final steps in alcohol synthesis, which are catalyzed by alcohol dehydrogenase and acetaldehyde CoA dehydrogenase. We have isolated a series of mutations affecting the expression of these enzymes. Some of these mutations are in the structural genes for these enzymes; others affect the regulation of the adh operon. We have recently cloned the genes coding for these enzymes and are now studying the effect of multiple copies of the adh gene on fermentative growth and its regulation. A recently invented technique, proton suicide has allowed the selection of a variety of novel mutants affecting fermentation which are presently being characterized. We have isolated a comprehensive collection of operon fusions in which the lacZ structural gene is fused to promoters that are inactive aerobically but active anaerobically. Although these genes (like adh) are only expressed under anaerobic conditions, the level of induction varies from two-fold to nearly 100-fold. The nitrogen source, medium pH, nature of the buffer, presence of alternative electron acceptors (e.g., nitrate), and other factors exert a great effect on the expression of many of these genes. In the near future we will investigate control mechanisms common to the adh operon and other anaerobically regulated genes.

  3. Antimicrobial-resistant Invasive Escherichia coli, Spain

    PubMed Central

    Oteo, Jesús; Lázaro, Edurne; de Abajo, Francisco J.; Baquero, Fernando; Campos, José

    2005-01-01

    To address the public health problem of antimicrobial resistance, the European Union founded the European Antimicrobial Resistance Surveillance System. A network of 32 Spanish hospitals, serving ≈9.6 million persons, submitted antimicrobial-susceptibility data on 7,098 invasive Escherichia coli species (2001–2003). Resistance to ampicillin, cotrimoxazole, ciprofloxacin, gentamicin, and tobramycin was found at rates of 59.9%, 32.6%, 19.3%, 6.8%, and 5.3%, respectively. Resistance to multiple drugs increased from 13.8% in 2001 to 20.6% in 2003 (p <0.0001). Antimicrobial consumption data were obtained from the Spanish National Health System. In spite of decreased cephalosporin and β-lactam use, overall extended-spectrum β-lactamase production increased from 1.6% (2001) to 4.1% (2003) (p <0.0001), mainly due to the rising prevalence of cefotaximases. Resistance to ciprofloxacin significantly increased, mostly in community-onset infections, which coincided with a rise in community quinolone use. Cotrimoxazole resistance remained stable at ≈30%, even though its use was dramatically reduced. PMID:15829192

  4. Genotoxicity of Graphene in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Sharma, Ananya

    Rapid advances in nanotechnology necessitate assessment of the safety of nanomaterials in the resulting products and applications. One key nanomaterial attracting much interest in many areas of science and technology is graphene. Graphene is a one atom thick carbon allotrope arranged in a two-dimensional honeycomb lattice. In addition to being extremely thin, graphene has several extraordinary physical properties such as its exceptional mechanical strength, thermal stability, and high electrical conductivity. Graphene itself is relatively chemically inert and therefore pristine graphene must undergo a process called functionalization, which is combination of chemical and physical treatments that change the properties of graphene, to make it chemically active. Functionalization of graphene is of crucial importance as the end application of graphene depends on proper functionalization. In the field of medicine, graphene is currently a nanomaterial of high interest for building biosensors, DNA transistors, and probes for cancer detection. Despite the promising applications of graphene in several areas of biomedicine, there have been only few studies in recent years that focus on evaluating cytotoxicity of graphene on cells, and almost no studies that investigate how graphene exposure affects cellular genetic material. Therefore, in this study we used a novel approach to evaluate the genotoxicity, i.e., the effects of graphene on DNA, using Escherichia coli as a prokaryotic model organism.

  5. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  6. Biochemistry of homologous recombination in Escherichia coli.

    PubMed Central

    Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

    1994-01-01

    Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

  7. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  8. Ribonuclease Sensitivity of Escherichia coli Ribosomes

    PubMed Central

    Santer, Melvin; Smith, Josephine R.

    1966-01-01

    Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099–1110. 1966.—The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10−4m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities. Images PMID:5332866

  9. Enterotoxigenic Escherichia coli Multilocus Sequence Types in Guatemala and Mexico

    PubMed Central

    Klena, John; Rodas, Claudia; Bourgeois, August Louis; Torres, Olga; Svennerholm, Ann-Mari; Sjöling, Åsa

    2010-01-01

    The genetic backgrounds of 24 enterotoxigenic Escherichia coli (ETEC) strains from Mexico and Guatemala expressing heat-stable toxin (ST) and coli surface antigen 6 (CS6) were analyzed. US travelers to these countries and resident children in Guatemala were infected by ETEC strains of sequence type 398, expressing STp and carrying genetically identical CS6 sequences. PMID:20031063

  10. Characterization of enterohemorrhagic Escherichia coli on veal hides and carcasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterohemorrhagic E. coli (EHEC) are Shiga toxin–producing Escherichia coli (STEC) associated with the most severe forms of foodborne illnesses. The United States Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) has identified a higher percentage of non-O157 EHEC compared to E....

  11. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

  12. Draft Genome Sequence of Uropathogenic Escherichia coli Strain NB8

    PubMed Central

    Mi, Zu-huang; Wang, Chun-xin; Zhu, Jian-ming

    2016-01-01

    Escherichia coli NB8 is a clinical pyelonephritis isolate. Here, we report the draft genome sequence of uropathogenic E. coli NB8, which contains drug resistance genes encoding resistance to beta-lactams, aminoglycosides, quinolones, macrolides, colistin, sulfonamide-trimethoprim, and tetracycline. NB8 infects the kidney and bladder, making it an important tool for studying E. coli pathogenesis. PMID:27609920

  13. Soil solarization reduces Escherichia coli O157:H7 and total Escherichia coli on cattle feedlot pen surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feedlot pen soils are a source for transmission of Escherichia coli O157:H7, and therefore a target for preharvest strategies to reduce this pathogen in cattle. The objective of this study was to determine the ability of soil solarization to reduce E. coli O157:H7 in feedlot surface material (FSM)....

  14. Free RNA polymerase in Escherichia coli.

    PubMed

    Patrick, Michael; Dennis, Patrick P; Ehrenberg, Mans; Bremer, Hans

    2015-12-01

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

  15. The Melibiose Transporter of Escherichia coli

    PubMed Central

    Fuerst, Oliver; Lin, Yibin; Granell, Meritxell; Leblanc, Gérard; Padrós, Esteve; Lórenz-Fonfría, Víctor A.; Cladera, Josep

    2015-01-01

    We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na+-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na+ and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200–330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na+ ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na+ ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na+ binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na+ binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions. PMID:25971963

  16. Novel Mechanism of Escherichia coli Porin Regulation

    PubMed Central

    Castillo-Keller, Maria; Vuong, Phu; Misra, Rajeev

    2006-01-01

    A novel mechanism of Escherichia coli porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated ipeX for inhibition of porin expression, and to DNA containing a 3′-truncated ompR gene. The major effect of ipeX on ompC expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced ompC transcription. ipeX was localized within an untranslated region of 247 base pairs between the stop codon of nmpC—a remnant porin gene from the cryptic phage qsr′ (DLP12) genome—and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from lc identical to that of ipeX. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the lc transcript, whose 3′ end contains the corresponding ipeX sequence, inhibits OmpC expression. Overexpression of ipeX RNA inhibited both OmpC and OmpF expression but not that of OmpA. ompC-phoA chimeric gene constructs revealed a 248-bp untranslated region of ompC required for ipeX-mediated inhibition. However, no sequence complementarity was found between ipeX and this region of ompC, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of ipeX on ompC, but not on ompF, was independent of the RNA chaperone Hfq. PMID:16385048

  17. Serogroups of Escherichia coli from drinking water.

    PubMed

    Ramteke, P W; Tewari, Suman

    2007-07-01

    Fifty seven isolates of thermotolerant E. coli were recovered from 188 drinking water sources, 45 (78.9%) were typable of which 15 (26.3%) were pathogenic serotypes. Pathogenic serogroup obtained were 04 (Uropathogenic E. coli, UPEC), 025 (Enterotoxigenic E. coli, ETEC), 086 (Enteropathogenic E. coli, EPEC), 0103 (Shiga-toxin producing E. coli, STEC), 0157 (Shiga-toxin producing E. coli, STEC), 08 (Enterotoxigenic E. coli, ETEC) and 0113 (Shiga-toxin producing E. coli, STEC). All the pathogenic serotypes showed resistance to bacitracin and multiple heavy metal ions. Resistance to streptomycin and cotrimazole was detected in two strains whereas resistance to cephaloridine, polymixin-B and ampicillin was detected in one strain each. Transfer of resistances to drugs and metallic ions was observed in 9 out of 12 strains studied. Resistances to bacitracin were transferred in all nine strains. Among heavy metals resistance to As(3+) followed by Cr(6+) were transferred more frequently.

  18. Environmental Escherichia coli: Ecology and public health implications - A review

    USGS Publications Warehouse

    Jang, Jeonghwan; Hur, Hor-Gil; Sadowsky, Michael J.; Byappanahalli, Muruleedhara; Yan, Tao; Ishii, Satoshi

    2017-01-01

    Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through feces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent fecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extra-intestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a fecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics provide the diversity and complexity of E. coli strains in various environments, affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments in regards to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.

  19. Investigation of ’Escherichia coli’ Enterotoxins

    DTIC Science & Technology

    1978-05-01

    E . coli diarrheal disease in man and domestic animals. Fundamentally, the design of the vaccine is based on the well- documented ability of cholera antitoxin to neutralize both cholera and heat- labile E . coli enterotoxins and on the ability of certain E . coli antigens to enhance the immune response to cholera toxoid and possibly whole-cell Cholera Vaccine, as

  20. Rapid Sterilization of Escherichia coli by Solution Plasma Process

    NASA Astrophysics Data System (ADS)

    Andreeva, Nina; Ishizaki, Takahiro; Baroch, Pavel; Saito, Nagahiro

    2012-12-01

    Solution plasma (SP), which is a discharge in the liquid phase, has the potential for rapid sterilization of water without chemical agents. The discharge showed a strong sterilization performance against Escherichia coli bacteria. The decimal value (D value) of the reduction time for E. coli by this system with an electrode distance of 1.0 mm was estimated to be approximately 1.0 min. Our discharge system in the liquid phase caused no physical damage to the E. coli and only a small increase in the temperature of the aqueous solution. The UV light generated by the discharge was an important factor in the sterilization of E. coli.

  1. Infection by verocytotoxin-producing Escherichia coli.

    PubMed Central

    Karmali, M A

    1989-01-01

    Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a newly recognized group of enteric pathogens which are increasingly being recognized as common causes of diarrhea in some geographic settings. Outbreak studies indicate that most patients with VTEC infection develop mild uncomplicated diarrhea. However, a significant risk of two serious and potentially life-threatening complications, hemorrhagic colitis and the hemolytic uremic syndrome, makes VTEC infection a public health problem of serious concern. The main reservoirs of VTEC appear to be the intestinal tracts of animals, and foods of animal (especially bovine) origin are probably the principal sources for human infection. The term VT refers to a family of subunit exotoxins with high biological activity. Individual VTEC strains elaborate one or both of at least two serologically distinct, bacteriophage-mediated VTs (VT1 and VT2) which are closely related to Shiga toxin and are thus also referred to as Shiga-like toxins. The holotoxins bind to cells, via their B subunits, to a specific receptor which is probably the glycolipid, globotriosyl ceramide (Gb3). Binding is followed by internalization of the A subunit, which, after it is proteolytically nicked and reduced to the A1 fragment, inhibits protein synthesis in mammalian cells by inactivating 60S ribosomal subunits through selective structural modification of 28S ribosomal ribonucleic acid. The mechanism of VTEC diarrhea is still controversial, and the relative roles of locally acting VT and "attaching and effacing adherence" of VTEC to the mucosa have yet to be resolved. There is increasing evidence that hemolytic uremic syndrome and possibly hemorrhagic colitis result from the systemic action of VT on vascular endothelial cells. The role of antitoxic immunity in preventing the systemic complications of VTEC infection is being explored. Antibiotics appear to be contraindicated in the treatment of VTEC infection. The most common VTEC serotype associated

  2. Intestinal Colonization by Enterotoxigenic Escherichia coli.

    DTIC Science & Technology

    1980-09-01

    E . coli is mediated by specific types of pili. These pili are antigenic and can be used in diagnosing enterotoxigenic E . coli infections. They are also good protective antigens. When pregnant dams are vaccinated parenterally or orally with pili on live piliated bacteria, they secrete antibodies against the pili in their milk. Neonates suckling dams so vaccinated are passively protected against fatal challenge by enterotoxigenic E . coli . Pili are also good candidate protective antigens for the development of vaccines to protect by

  3. Transcription of foreign DNA in Escherichia coli.

    PubMed

    Warren, René L; Freeman, John D; Levesque, Roger C; Smailus, Duane E; Flibotte, Stephane; Holt, Robert A

    2008-11-01

    Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli sigma(70) (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes.

  4. Recurrent Hemolytic and Uremic Syndrome Induced by Escherichia Coli

    PubMed Central

    Commereuc, Morgane; Weill, Francois-Xavier; Loukiadis, Estelle; Gouali, Malika; Gleizal, Audrey; Kormann, Raphaël; Ridel, Christophe; Frémeaux-Bacchi, Véronique; Rondeau, Eric; Hertig, Alexandre

    2016-01-01

    Abstract A widespread belief is that typical hemolytic and uremic syndrome (HUS) does not recur. We report the case of a patient infected twice with raw milk taken from his own cow and containing a Shiga toxin–producing Escherichia coli O174:H21 that induced recurrent HUS causing severe renal and cerebral disorders. A genomic comparison of the human and bovine Shiga toxin–producing Escherichia coli O174:H21 isolates revealed that they were identical. Typical HUS may recur. Since milk from this animal was occasionally distributed locally, thereby posing a serious threat for the whole village, this particular cow was destroyed. PMID:26735524

  5. [Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli].

    PubMed

    Ptitsyn, L R; Fatova, M A; Stepanov, A I

    1990-02-01

    Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.

  6. Diarrheagenic Escherichia coli in Children from Costa Rica

    PubMed Central

    Pérez, Cristian; Gómez-Duarte, Oscar G.; Arias, María L.

    2010-01-01

    More than 5,000 diarrheal cases per year receive medical care at the National Children's Hospital of Costa Rica, and nearly 5% of them require hospitalization. A total of 173 Escherichia coli strains isolated from children with diarrhea were characterized at the molecular, serologic, and phenotypic level. Multiplex and duplex polymerase chain reactions were used to detect the six categories of diarrheagenic E. coli. Thirty percent (n = 52) of the strains were positive, indicating a high prevalence among the pediatric population. Enteropathogenic E. coli and enteroinvasive E. coli pathotypes were the most prevalent (21% and 19%, respectively). Pathogenic strains were distributed among the four E. coli phylogenetic groups A, B1, B2, and D, with groups A and B1 the most commonly found. This study used molecular typing to evaluate the prevalence of diarrheagenic E. coli reported in Costa Rica and demonstrated the importance of these pathotypes in the pediatric population. PMID:20682870

  7. Large Surface Blebs on Escherichia coli Heated to Inactivating Temperatures

    PubMed Central

    Scheie, Paul; Ehrenspeck, Susan

    1973-01-01

    Large surface blebs were observed with phase-contrast optics on Escherichia coli B/r and Bs-1 heated to temperatures at which colony-forming ability was lost. Characterization of such blebs was consistent with the view that they were formed by a physical process and were bounded by the outer membrane of the cell. A hypothesis for thermal inactivation of E. coli is presented that places membrane damage near the primary lethal event. Images PMID:4196258

  8. Expression of staphylococcal enterotoxin C1 in Escherichia coli.

    PubMed Central

    Bohach, G A; Schlievert, P M

    1987-01-01

    The structural gene encoding staphylococcal enterotoxin C1 was cloned into Escherichia coli and localized on a 1.5-kilobase HindIII-ClaI DNA fragment by subcloning. The toxin was partially purified from E. coli clones and shown to be immunologically identical to enterotoxin C1 from Staphylococcus aureus. The cloned toxin also had the same molecular weight (26,000) and charge heterogeneity as staphylococcus-derived enterotoxin. Toxins from both sources were equally biologically active. Images PMID:3542834

  9. 76 FR 72331 - Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-23

    ... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... methods for controlling non-O157 Shiga toxin-producing Escherichia coli in raw, intact and non-intact beef... Escherichia coli in raw, intact and non-intact beef products and product components on or before December...

  10. Multidrug-resistant Escherichia coli in Asia: epidemiology and management.

    PubMed

    Sidjabat, Hanna E; Paterson, David L

    2015-05-01

    Escherichia coli has become multiresistant by way of production of a variety of β-lactamases. The prevalence of CTX-M-producing E. coli has reached 60-79% in certain parts of Asia. The acquisition of CTX-M plasmids by E. coli sequence type 131, a successful clone of E. coli, has caused further dissemination of CTX-M-producing E. coli. The prevalence of carbapenemase-producing E. coli, especially Klebsiella pneumoniae carbapenemase, and New Delhi metallo-β-lactamase (NDM)-producing E. coli has been increasing in Asia. K. pneumoniae carbapenemase and NDM have now been found in E. coli sequence type 131. The occurrence of NDM-producing E. coli is a major concern particularly in the Indian subcontinent, but now elsewhere in Asia as well. There are multiple reasons why antibiotic resistance in E. coli in Asia has reached such extreme levels. Approaches beyond antibiotic therapy, such as prevention of antibiotic resistance by antibiotic stewardship and protecting natural microbiome, are strategies to avoid further spread of antibiotic resistance.

  11. Short-Chain Chromate Ion Transporter Proteins from Bacillus subtilis Confer Chromate Resistance in Escherichia coli▿

    PubMed Central

    Díaz-Magaña, Amada; Aguilar-Barajas, Esther; Moreno-Sánchez, Rafael; Ramírez-Díaz, Martha I.; Riveros-Rosas, Héctor; Vargas, Eréndira; Cervantes, Carlos

    2009-01-01

    Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily (ywrB and ywrA) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C, respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli, suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm. PMID:19581367

  12. The quantitative and condition-dependent Escherichia coli proteome

    PubMed Central

    Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

    2016-01-01

    Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

  13. An integrated database to support research on Escherichia coli

    SciTech Connect

    Baehr, A.; Dunham, G.; Matsuda, Hideo; Michaels, G.; Taylor, R.; Overbeek, R.; Rudd, K.E.; Ginsburg, A.; Joerg, D.; Kazic, T.; Hagstrom, R.; Zawada, D.; Smith, C.; Yoshida, Kaoru

    1992-01-01

    We have used logic programming to design and implement a prototype database of genomic information for the model bacterial organism Escherichia coli. This report presents the fundamental database primitives that can be used to access and manipulate data relating to the E. coli genome. The present system, combined with a tutorial manual, provides immediate access to the integrated knowledge base for E. coli chromosome data. It also serves as the foundation for development of more user-friendly interfaces that have the same retrieval power and high-level tools to analyze complex chromosome organization.

  14. YeeO from Escherichia coli exports flavins.

    PubMed

    McAnulty, Michael J; Wood, Thomas K

    2014-01-01

    Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters.

  15. Heat-stable Escherichia coli enterotoxin production in vivo.

    PubMed Central

    Whipp, S C; Moon, H W; Lyon, N C

    1975-01-01

    Hysterectomy-derived, colostrum-deprived piglets were infected with enterotoxigenic Escherichia coli on day 4 of life. Samples of feces and intestinal contents were collected and tested in infant mice for enterotoxic activity. Positive enterotoxic responses were observed in mice given filtrates of feces and intestinal contents from piglets infected withe enterotoxigenic E. coli known to produce heat-stable enterotoxin but not heat-liabile enterotoxin in vitro. It is concluded that heat-stable enterotoxigenic E. coli induce diarrhea by production of heat-stable enterotoxin in vivo. PMID:1097335

  16. Reassessing Escherichia coli as a cell factory for biofuel production.

    PubMed

    Wang, Chonglong; Pfleger, Brian F; Kim, Seon-Won

    2017-03-11

    Via metabolic engineering, industrial microorganisms have the potential to convert renewable substrates into a wide range of biofuels that can address energy security and environmental challenges associated with current fossil fuels. The user-friendly bacterium, Escherichia coli, remains one of the most frequently used hosts for demonstrating production of biofuel candidates including alcohol-, fatty acid- and terpenoid-based biofuels. In this review, we summarize the metabolic pathways for synthesis of these biofuels and assess enabling technologies that assist in regulating biofuel synthesis pathways and rapidly assembling novel E. coli strains. These advances maintain E. coli's position as a prominent host for developing cell factories for biofuel production.

  17. Enteropathogenic Escherichia coli Serotypes and Endemic Diarrhea in Infants

    PubMed Central

    Toledo, M. Regina F.; Alvariza, M. do Carmo B.; Murahovschi, Jayme; Ramos, Sonia R. T. S.; Trabulsi, Luiz R.

    1983-01-01

    Enteropathogenic Escherichia coli serotypes were searched for in feces of 550 children with endemic diarrhea and in 129 controls, in São Paulo, in 1978 and 1979; serotypes O111ab:H−, O111ab:H2, and O119:H6 were significantly associated with diarrhea in children 0 to 5 months old and were the most frequent agents of diarrhea in this age group as compared with enterotoxigenic and enteroinvasive E. coli, Salmonella sp., Shigella sp., and Yersinia enterocolitica. It is concluded that various enteropathogenic E. coli serotypes may be agents of endemic infantile diarrhea. PMID:6339384

  18. Sources of Escherichia coli in a Coastal Subtropical Environment

    PubMed Central

    Solo-Gabriele, Helena M.; Wolfert, Melinda A.; Desmarais, Timothy R.; Palmer, Carol J.

    2000-01-01

    Sources of Escherichia coli in a coastal waterway located in Ft. Lauderdale, Fla., were evaluated. The study consisted of an extensive program of field measurements designed to capture spatial and temporal variations in E. coli concentrations as well as experiments conducted under laboratory-controlled conditions. E. coli from environmental samples was enumerated by using a defined substrate technology (Colilert-18). Field sampling tasks included sampling the length of the North Fork to identify the river reach contributing high E. coli levels, autosampler experiments at two locations, and spatially intense sampling efforts at hot spots. Laboratory experiments were designed to simulate tidal conditions within the riverbank soils. The results showed that E. coli entered the river in a large pulse during storm conditions. After the storm, E. coli levels returned to baseline levels and varied in a cyclical pattern which correlated with tidal cycles. The highest concentrations were observed during high tide, whereas the lowest were observed at low tide. This peculiar pattern of E. coli concentrations between storm events was caused by the growth of E. coli within riverbank soils which were subsequently washed in during high tide. Laboratory analysis of soil collected from the riverbanks showed increases of several orders of magnitude in soil E. coli concentrations. The ability of E. coli to multiply in the soil was found to be a function of soil moisture content, presumably due to the ability of E. coli to outcompete predators in relatively dry soil. The importance of soil moisture in regulating the multiplication of E. coli was found to be critical in tidally influenced areas due to periodic wetting and drying of soils in contact with water bodies. Given the potential for growth in such systems, E. coli concentrations can be artificially elevated above that expected from fecal impacts alone. Such results challenge the use of E. coli as a suitable indicator of water

  19. Structure of Water in Escherichia Coli B

    DTIC Science & Technology

    structure broadening of the NMR water spectrum. Using bacteria grown in the special chemically defined medium, we showed that the water in E. coli B was highly ordered and was very different from ’free’ water and from polywater .

  20. Slugs: Potential Novel Vectors of Escherichia coli O157

    PubMed Central

    Sproston, Emma L.; Macrae, M.; Ogden, Iain D.; Wilson, Michael J.; Strachan, Norval J. C.

    2006-01-01

    Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157. PMID:16391036

  1. Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004.

    PubMed

    Qadri, Firdausi; Khan, Ashraful I; Faruque, Abu Syed G; Begum, Yasmin Ara; Chowdhury, Fahima; Nair, Gopinath B; Salam, Mohammed A; Sack, David A; Svennerholm, Ann-Mari

    2005-07-01

    Flooding in Dhaka in July 2004 caused epidemics of diarrhea. Enterotoxigenic Escherichia coli (ETEC) was almost as prevalent as Vibrio cholerae O1 in diarrheal stools. ETEC that produced heat-stable enterotoxin alone was most prevalent, and 78% of strains had colonization factors. Like V. cholerae O1, ETEC can cause epidemic diarrhea.

  2. armA and aminoglycoside resistance in Escherichia coli.

    PubMed

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C; Moreno, Miguel A; Courvalin, Patrice; Domínguez, Lucas

    2005-06-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

  3. armA and Aminoglycoside Resistance in Escherichia coli

    PubMed Central

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C.; Courvalin, Patrice; Domínguez, Lucas

    2005-01-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant. PMID:15963296

  4. Escherichia coli as other Enterobacteriaceae: food poisoning and health effects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many Escherichia coli strains are harmless, and they are an important commensal in the intestinal microflora; however, pathogenic strains also exist. The pathogenic strains can be divided into diarrhea-inducing strains and strains that reside in the intestines but only cause disease in bodily sites...

  5. Escherichia coli growth studied by dual-parameter flow cytophotometry.

    PubMed Central

    Steen, H B; Boye, E

    1981-01-01

    The growth of Escherichia coli cells has been analyzed for the first time by dual-parameter flow cytophotometry, in which the deoxyribonucleic acid and protein contents of single bacteria have been measured simultaneously with an accuracy of a few percent and at a rate of 3,000 cells/s. PMID:7007339

  6. More than a locomotive organelle: flagella in Escherichia coli.

    PubMed

    Zhou, Mingxu; Yang, Yang; Chen, Panlin; Hu, Huijie; Hardwidge, Philip R; Zhu, Guoqiang

    2015-11-01

    The flagellum is a locomotive organelle that allows bacteria to respond to chemical gradients. This review summarizes the current knowledge regarding Escherichia coli flagellin variants and the role of flagella in bacterial functions other than motility, including the relationship between flagella and bacterial virulence.

  7. Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332.

    PubMed

    Saldaña-Ahuactzi, Zeus; Cruz-Córdova, Ariadnna; Rodea, Gerardo E; Porta, Helena; Navarro-Ocaña, Armando; Eslava-Campos, Carlos; Cevallos, Miguel A; Xicohtencatl-Cortes, Juan

    2017-02-23

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México.

  8. Stringent control of FLP recombinase in Escherichia coli.

    PubMed

    Bowden, Steven D; Palani, Nagendra P; Libourel, Igor G L

    2017-02-01

    Site specific recombinases are invaluable tools in molecular biology, and are emerging as powerful recorders of cellular events in synthetic biology. We have developed a stringently controlled FLP recombinase system in Escherichia coli using an arabinose inducible promoter combined with a weak ribosome binding site.

  9. Enteroinvasive Escherichia coli severe dysentery complicated by rotavirus gastroenteritis.

    PubMed

    Pacheco-Gil, Leova; Ochoa, Theresa J; Flores-Romo, Leopoldo; DuPont, Herbert L; Estrada-Garcia, Teresa

    2006-11-01

    Enteroinvasive Escherichia coli (EIEC) is an important agent of pediatric diarrhea and dysentery in developing countries. We report a life-threatening severe dysentery case due to EIEC in a malnourished 4-month-old male, native Indian infant co-infected with rotavirus. The severe gastrointestinal bleeding anemia and hypovolemic shock was successfully treated with IV blood transfusions, rehydration and antibiotic therapy.

  10. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    The increase in resistance rates to trimehtoprim-sulfamethoxazole (TMP/SMX) in isolates of Escherichia coli has become a matter of increasing concern. This has been particularly true in reference to community acquired urinary tract infections (UTI). This study utilized sewage i...

  11. Escherichia coli and other Enterobacteriaceae: Food poisoning and health effects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The family Enterobactericeae consists of rod-shaped, Gram-negative, facultatively anaerobic, non-spore forming bacteria and also includes the food-borne pathogens, Cronobacter spp., Escherichia coli, Salmonella enterica, Shigella spp., and Yersinia spp. Illness caused by these pathogens is acquired...

  12. Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332

    PubMed Central

    Saldaña-Ahuactzi, Zeus; Cruz-Córdova, Ariadnna; Rodea, Gerardo E.; Porta, Helena; Navarro-Ocaña, Armando; Eslava-Campos, Carlos

    2017-01-01

    ABSTRACT   Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México. PMID:28232434

  13. New types of Escherichia coli recombination-deficient mutants.

    PubMed

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  14. New types of Escherichia coli recombination-deficient mutants.

    PubMed Central

    Freifelder, D

    1976-01-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency. PMID:789362

  15. Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

    EPA Science Inventory

    Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfe...

  16. rRNA transcription rate in Escherichia coli.

    PubMed Central

    Gotta, S L; Miller, O L; French, S L

    1991-01-01

    The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s. Images FIG. 1 PMID:1717439

  17. Multidrug-Resistant Escherichia coli in Bovine Animals, Europe

    PubMed Central

    Brennan, Evan; Martins, Marta; McCusker, Matthew P.; Wang, Juan; Alves, Bruno Martins; Hurley, Daniel; El Garch, Farid; Woehrlé, Frédérique; Miossec, Christine; McGrath, Leisha; Srikumar, Shabarinath; Wall, Patrick

    2016-01-01

    Of 150 Escherichia coli strains we cultured from specimens taken from cattle in Europe, 3 had elevated MICs against colistin. We assessed all 3 strains for the presence of the plasmid-mediated mcr-1 gene and identified 1 isolate as mcr-1–positive and co-resistant to β-lactam, florfenicol, and fluoroquinolone antimicrobial compounds. PMID:27533105

  18. EcoCyc: Encyclopedia of Escherichia coli genes and metabolism.

    PubMed

    Karp, P D; Riley, M; Paley, S M; Pellegrini-Toole, A; Krummenacker, M

    1998-01-01

    The encyclopedia of Escherichia coli genes and metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of E.coli. The database describes 3030 genes of E.coli , 695 enzymes encoded by a subset of these genes, 595 metabolic reactions that occur in E.coli, and the organization of these reactions into 123 metabolic pathways. The EcoCyc graphical user interface allows scientists to query and explore the EcoCyc database using visualization tools such as genomic-map browsers and automatic layouts of metabolic pathways. EcoCyc can be thought of as an electronic review article because of its copious references to the primary literature, and as a (qualitative) computational model of E.coli metabolism. EcoCyc is available at URL http://ecocyc.PangeaSystems.com/ecocyc/

  19. Phylogenetic Group Determination of Escherichia coli Isolated from Animals Samples

    PubMed Central

    Morcatti Coura, Fernanda; Diniz, Soraia de Araújo; Silva, Marcos Xavier; Mussi, Jamili Maria Suhet; Barbosa, Silvia Minharro; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2015-01-01

    This study analyzes the occurrence and distribution of phylogenetic groups of 391 strains of Escherichia coli isolated from poultry, cattle, and water buffalo. The frequency of the phylogroups was A = 19%, B1 = 57%, B2 = 2.3%, C = 4.6%, D = 2.8%, E = 11%, and F = 3.3%. Phylogroups A (P < 0.001) and F (P = 0.018) were associated with E. coli strains isolated from poultry, phylogroups B1 (P < 0.001) and E (P = 0.002) were associated with E. coli isolated from cattle, and phylogroups B2 (P = 0.003) and D (P = 0.017) were associated with E. coli isolated from water buffalo. This report demonstrated that some phylogroups are associated with the host analyzed and the results provide knowledge of the phylogenetic composition of E. coli from domestic animals. PMID:26421310

  20. Glycerol elicits energy taxis of Escherichia coli and Salmonella typhimurium.

    PubMed

    Zhulin, I B; Rowsell, E H; Johnson, M S; Taylor, B L

    1997-05-01

    Escherichia coli and Salmonella typhimurium show positive chemotaxis to glycerol, a chemical previously reported to be a repellent for E. coli. The threshold of the attractant response in both species was 10(-6) M glycerol. Glycerol chemotaxis was energy dependent and coincident with an increase in membrane potential. Metabolism of glycerol was required for chemotaxis, and when lactate was present to maintain energy production in the absence of glycerol, the increases in membrane potential and chemotactic response upon addition of glycerol were abolished. Methylation of a chemotaxis receptor was not required for positive glycerol chemotaxis in E. coli or S. typhimurium but is involved in the negative chemotaxis of E. coli to high concentrations of glycerol. We propose that positive chemotaxis to glycerol in E. coli and S. typhimurium is an example of energy taxis mediated via a signal transduction pathway that responds to changes in the cellular energy level.

  1. Polyerositis and Arthritis Due to Escherichia coli in Gnotobiotic Pigs

    PubMed Central

    Waxler, G. L.; Britt, A. L.

    1972-01-01

    Forty gnotobiotic pigs from six litters were exposed orally to Escherichia coli 083:K·:NM at 69 to 148 hours of age, while 17 pigs from the same litters served as unexposed controls. Clinical signs of infection included fever, anorexia, diarrhea, lameness, and reluctance to move. Eighty-four percent of the exposed pigs in four litters died, while only 13% in two litters died. Gross and microscopic lesions included serofibrinous to fibrinopurulent polyserositis in 96% of the exposed pigs in four litters and 33% of the exposed pigs in two litters. A few pigs had gross and/or microscopic lesions of arthritis. Escherichia coli was routinely isolated from the serous and synovial cavities of infected pigs. Anti-hog cholera serum administered orally as a colostrum substitute gave partial protection against E. coli infection. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8. PMID:4261837

  2. Cytotoxic Escherichia coli strains encoding colibactin colonize laboratory mice.

    PubMed

    García, Alexis; Mannion, Anthony; Feng, Yan; Madden, Carolyn M; Bakthavatchalu, Vasudevan; Shen, Zeli; Ge, Zhongming; Fox, James G

    2016-12-01

    Escherichia coli strains have not been fully characterized in laboratory mice and are not currently excluded from mouse colonies. Colibactin (Clb), a cytotoxin, has been associated with inflammation and cancer in humans and animals. We performed bacterial cultures utilizing rectal swab, fecal, and extra intestinal samples from clinically unaffected or affected laboratory mice. Fifty-one E. coli were isolated from 45 laboratory mice, identified biochemically, and selected isolates were serotyped. The 16S rRNA gene was amplified and sequenced for specific isolates, PCR used for clbA and clbQ gene amplification, and phylogenetic group identification was performed on all 51 E. coli strains. Clb genes were sequenced and selected E. coli isolates were characterized using a HeLa cell cytotoxicity assay. Forty-five of the 51 E. coli isolates (88%) encoded clbA and clbQ and belonged to phylogenetic group B2. Mouse E. coli serotypes included: O2:H6, O-:H-, OM:H+, and O22:H-. Clb-encoding O2: H6 mouse E. coli isolates were cytotoxic in vitro. A Clb-encoding E. coli was isolated from a clinically affected genetically modified mouse with cystic endometrial hyperplasia. Our findings suggest that Clb-encoding E. coli colonize laboratory mice and may induce clinical and subclinical diseases that may impact experimental mouse models.

  3. Using zebra mussels to monitor Escherichia coli in environmental waters.

    PubMed

    Selegean, J P; Kusserow, R; Patel, R; Heidtke, T M; Ram, J L

    2001-01-01

    Use of the zebra mussel (Dreissena polymorpha) as an indicator of previously elevated bacteria concentrations in a watershed was examined. The ability of the zebra mussel to accumulate and purge Escherichia coli over several days was investigated in both laboratory and field experiments. In laboratory experiments, periodic enumeration of E. coli in mussels that had been exposed to a dilute solution of raw sewage demonstrated that (i) maximum concentrations of E. coli are reached within a few hours of exposure to sewage, (ii) the tissue concentration attained is higher than the concentration in the ambient water, and (iii) the E. coli concentrations take several days to return to preexposure concentrations when mussels are subsequently placed in sterile water. In field experiments conducted in southeast Michigan in the Clinton River watershed, brief increases in E. coli concentrations in the water were accompanied by increases in mussel concentrations of E. coli that lasted 2 or 3 d. The ability of mussels to retain and to concentrate E. coli made it possible to detect E. coli in the environment under conditions that conventional monitoring may often miss. Sampling caged mussels in a river and its tributaries may enable watershed managers to reduce the sampling frequency normally required to identify critical E. coli sources, thereby providing a more cost-effective river monitoring strategy for bacterial contamination.

  4. Lytic bacteriophages reduce Escherichia coli O157

    PubMed Central

    Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

    2013-01-01

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce. PMID:23819106

  5. Production of itaconic acid in Escherichia coli expressing recombinant α-amylase using starch as substrate.

    PubMed

    Okamoto, Shusuke; Chin, Taejun; Nagata, Keisuke; Takahashi, Tetsuya; Ohara, Hitomi; Aso, Yuji

    2015-05-01

    Several studies on fermentative production of a vinyl monomer itaconic acid from hydrolyzed starch using Aspergillus terreus have been reported. Herein, we report itaconic acid production by Escherichia coli expressing recombinant α-amylase, using soluble starch as its sole carbon source. To express α-amylase in E. coli, we first constructed recombinant plasmids expressing α-amylases by using cell surface display technology derived from two amylolytic bacteria, Bacillus amyloliquefaciens NBRC 15535(T) and Streptococcus bovis NRIC 1535. The recombinant α-amylase from S. bovis (SBA) showed activity at 28°C, which is the optimal temperature for production of itaconic acid, while α-amylase from B. amyloliquefaciens displayed no noticeable activity. E. coli cells expressing SBA produced 0.15 g/L itaconic acid after 69 h cultivation under pH-stat conditions, using 1% starch as the sole carbon source. In fact, E. coli cells expressing SBA had similar growth rates when grown in the presence of 1% glucose or starch, thereby highlighting the expression of an active α-amylase that enabled utilization of starch to produce itaconic acid in E. coli.

  6. Study of the effects of high-energy proton beams on escherichia coli

    NASA Astrophysics Data System (ADS)

    Park, Jeong Chan; Jung, Myung-Hwan

    2015-10-01

    Antibiotic-resistant bacterial infection is one of the most serious risks to public health care today. However, discouragingly, the development of new antibiotics has progressed little over the last decade. There is an urgent need for alternative approaches to treat antibiotic-resistant bacteria. Novel methods, which include photothermal therapy based on gold nano-materials and ionizing radiation such as X-rays and gamma rays, have been reported. Studies of the effects of high-energy proton radiation on bacteria have mainly focused on Bacillus species and its spores. The effect of proton beams on Escherichia coli (E. coli) has been limitedly reported. Escherichia coli is an important biological tool to obtain metabolic and genetic information and is a common model microorganism for studying toxicity and antimicrobial activity. In addition, E. coli is a common bacterium in the intestinal tract of mammals. In this research, the morphological and the physiological changes of E. coli after proton irradiation were investigated. Diluted solutions of cells were used for proton beam radiation. LB agar plates were used to count the number of colonies formed. The growth profile of the cells was monitored by using the optical density at 600 nm. The morphology of the irradiated cells was observed with an optical microscope. A microarray analysis was performed to examine the gene expression changes between irradiated samples and control samples without irradiation. E coli cells have observed to be elongated after proton irradiation with doses ranging from 13 to 93 Gy. Twenty-two were up-regulated more than twofold in proton-irradiated samples (93 Gy) compared with unexposed one.

  7. Experimental Escherichia coli O157:H7 carriage in calves.

    PubMed Central

    Brown, C A; Harmon, B G; Zhao, T; Doyle, M P

    1997-01-01

    Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding. PMID:8979335

  8. Travelers' diarrhea and toxigenic Escherichia coli.

    PubMed

    Gorbach, S L; Kean, B H; Evans, D G; Evans, D J; Bessudo, D

    1975-05-01

    In a group of 133 United States students studied for 18 days after arriving in Mexico, diarrhea developed in 38 (29 per cent). Diarrhea rarely began before the fourth day, and the mean onset was 13 days after arrival. Symptoms lasted an average of 3.4 days but persisted in 21 per cent of sick students. Heat-labile enterotoxin-producing Escheria coli was found in the stools of 72 per cent of sick and 15 per cent of healthy students. None had heat-labile Esch. coli when they entered Mexico. The incubation period was short, generally 24 to 48 hours, and the carrier state was five days or less in 82 per cent of students surveyed. Entamoeba histolytica was found in 6 per cent of cases of diarrhea, but not salmonella, shigella or penetrating Esch. coli. These studies suggest that approximately 70 per cent of travelers' diarrhea in Mexico is associated with heat-labile toxigenic strains of Esch. coli.

  9. Genomic Comparative Study of Bovine Mastitis Escherichia coli

    PubMed Central

    Kempf, Florent; Slugocki, Cindy; Blum, Shlomo E.; Leitner, Gabriel; Germon, Pierre

    2016-01-01

    Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes. PMID:26809117

  10. [Acute diarrheal disease caused by enteropathogenic Escherichia coli in Colombia].

    PubMed

    Gómez-Duarte, Oscar G

    2014-10-01

    Intestinal Escherichia coli pathogens are leading causes of acute diarrheal disease in children less than 5 years in Latin America, Africa and Asia and a leading cause of death in children living in poorest communities in Africa and South East Asia. Studies on the role of E. coli pathogens in childhood diarrhea in Colombia and other countries in Latin America are limited due to the lack of detection assays in clinical laboratories at the main urban medical centers. Recent studies report that enterotoxigenic E. coli is the most common E. coli pathogens associated with diarrhea in children less than 5 years of age. Other E. coli pathotypes have been detected in children with diarrhea including enteropathogenic, enteroaggregative, shiga-toxin producing and diffusely adherent E. coli. It was also found that meat and vegetables at retail stores are contaminated with Shiga-toxin producing E. coli and enteroaggregative E. coli, suggesting that food products are involved in transmission and infection of the susceptible host. More studies are necessary to evaluate the mechanisms of transmission, the impact on the epidemiology of diarrheal disease, and management strategies and prevention of these pathogens affecting the pediatric population in Colombia.

  11. Proton-linked D-xylose transport in Escherichia coli.

    PubMed Central

    Lam, V M; Daruwalla, K R; Henderson, P J; Jones-Mortimer, M C

    1980-01-01

    The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli. PMID:6995439

  12. Biosynthesis of Two Flavones, Apigenin and Genkwanin, in Escherichia coli.

    PubMed

    Lee, Hyejin; Kim, Bong Gyu; Kim, Mihyang; Ahn, Joong-Hoon

    2015-09-01

    The flavonoid apigenin and its O-methyl derivative, genkwanin, have various biological activities and can be sourced from some vegetables and fruits. Microorganisms are an alternative for the synthesis of flavonoids. Here, to synthesize genkwanin from tyrosine, we first synthesized apigenin from p-coumaric acid using four genes (4CL, CHS, CHI, and FNS) in Escherichia coli. After optimization of different combinations of constructs, the yield of apigenin was increased from 13 mg/l to 30 mg/l. By introducing two additional genes (TAL and POMT7) into an apigenin-producing E. coli strain, we were able to synthesize 7-O-methyl apigenin (genkwanin) from tyrosine. In addition, the tyrosine content in E. coli was modulated by overexpressing aroG and tyrA. The engineered E. coli strain synthesized approximately 41 mg/l genkwanin.

  13. [Escherichia coli R live vaccine Suicolplex "Dessau"].

    PubMed

    Michael-Meese, M; Klie, H; Schöll, W

    1980-01-01

    Immunisation of pregnant sows prior to parturition has long proved to be a good method to forestall coli dysentery in piglets before weaning. Inactivated vaccines of the pathogenetically important E. coli serogroups with and without adjuvant so far were primarily used at international level. A vaccine of that kind has become available in the GDR more than eight years ago. Its name is Coliporc "Dessau". A live vaccine has been developed from two R-mutants at the authors' institute. The effectiveness of that live vaccine on laboratory animals and in field experiments is reported in this paper together with possibilities of differential diagnosis to distinguish wild strains from the mutants. The live vaccine was commercially registered under the name of Suicolpex "Dessau", in spring 1976.

  14. Compilation of DNA sequences of Escherichia coli

    PubMed Central

    Kröger, Manfred

    1989-01-01

    We have compiled the DNA sequence data for E.coli K12 available from the GENBANK and EMBO databases and over a period of several years independently from the literature. We have introduced all available genetic map data and have arranged the sequences accordingly. As far as possible the overlaps are deleted and a total of 940,449 individual bp is found to be determined till the beginning of 1989. This corresponds to a total of 19.92% of the entire E.coli chromosome consisting of about 4,720 kbp. This number may actually be higher by some extra 2% derived from the sequence of lysogenic bacteriophage lambda and the various insertion sequences. This compilation may be available in machine readable form from one of the international databanks in some future. PMID:2654890

  15. Genotypic Characterization of Egypt Enterotoxigenic Escherichia coli Isolates Expressing Coli Surface Antigen 6

    DTIC Science & Technology

    2013-02-01

    USA Abstract Introduction: One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on...results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface...has received considerable attention as a target for vaccine development [11-14]. CS6 is immunogenic in humans both after natural infection and

  16. Metabolic engineering of Escherichia coli for 1-butanol production.

    PubMed

    Atsumi, Shota; Cann, Anthony F; Connor, Michael R; Shen, Claire R; Smith, Kevin M; Brynildsen, Mark P; Chou, Katherine J Y; Hanai, Taizo; Liao, James C

    2008-11-01

    Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.

  17. Functional role of bdm during flagella biogenesis in Escherichia coli.

    PubMed

    Kim, Ji-Sun; Kim, Yu Jin; Seo, Sojin; Seong, Maeng-Je; Lee, Kangseok

    2015-03-01

    The biofilm-dependent modulation gene (bdm) has recently been shown to play a role in osmotic-induced formation of biofilm in Escherichia coli. In this study, we demonstrated that deletion of bdm results in down-regulation of flagella biosynthesis genes and, consequently, a defect in E. coli motility. In addition, we employed atomic force microscopy to confirm the absence of flagella-like structures on the surface of bdm-null cells. These findings indicate that bdm plays a key role in regulatory pathway for the formation of flagella.

  18. PROPERTIES OF A BACTERIOPHAGE DERIVED FROM ESCHERICHIA COLI K235

    PubMed Central

    Jesaitis, Margeris A.; Hutton, John J.

    1963-01-01

    A temperate bacteriophage was isolated from the colicinogenic strain of Escherichia coli K235 and characterized. This phage, termed PK, is related to P2 virus morphologically, serologically, and, possibly, genetically and it bears no relationship to the T-even phages. It was also demonstrated that PK virus and colicine K differ both in their host range and in their immunological specificity, and that PK prophage does not induce the colicinogenesis in its host bacterium. It was concluded that the formation of colicine K. and PK phage in E. coli K235 are controlled by different genetic determinants. PMID:14029160

  19. Nitric oxide donor-mediated killing of bioluminescent Escherichia coli.

    PubMed Central

    Virta, M; Karp, M; Vuorinen, P

    1994-01-01

    The antimicrobial activities of two nitric oxide-releasing compounds against Escherichia coli were investigated by using recombinant E. coli cloned with a luciferase gene from Pyrophorus plagiophthalamus. Since luciferase uses intracellular ATP to generate visible light which can be measured from living cells in real time, we wanted to compare the extent to which cell viability parallels light emission. Results from luminescence measurements and CFU counts were in good agreement, and the decrease in light emission was shown to provide a rapid and more sensitive indication of cytotoxicity. PMID:7695261

  20. Accelerated glycerol fermentation in Escherichia coli using methanogenic formate consumption.

    PubMed

    Richter, Katrin; Gescher, Johannes

    2014-06-01

    Escherichia coli can ferment glycerol anaerobically only under very defined restrictive conditions. Hence, it was the aim of this study to overcome this limitation via a co-cultivation approach. Anaerobic glycerol fermentation by a pure E. coli culture was compared to a co-culture that also contained the formate-oxidizing methanogen Methanobacterium formicicum. Co-cultivation of the two strains led to a more than 11-fold increased glycerol consumption. Furthermore, it supported a constantly neutral pH and a shift from ethanol to succinate production. Moreover, M. formicicum was analyzed for its ability to grow on different standard media and a surprising versatility could be demonstrated.

  1. Bacterial self-defence: how Escherichia coli evades serum killing.

    PubMed

    Miajlovic, Helen; Smith, Stephen G

    2014-05-01

    The ability to survive the bactericidal action of serum is advantageous to extraintestinal pathogenic Escherichia coli that gain access to the bloodstream. Evasion of the innate defences present in serum, including complement and antimicrobial peptides, involves multiple factors. Serum resistance mechanisms utilized by E. coli include the production of protective extracellular polysaccharide capsules and expression of factors that inhibit or interfere with the complement cascade. Recent studies have also highlighted the importance of structural integrity of the cell envelope in serum survival. These survival strategies are outlined in this review with particular attention to novel findings and recent insights into well-established resistance mechanisms.

  2. Escherichia coli as a model active colloid: A practical introduction.

    PubMed

    Schwarz-Linek, Jana; Arlt, Jochen; Jepson, Alys; Dawson, Angela; Vissers, Teun; Miroli, Dario; Pilizota, Teuta; Martinez, Vincent A; Poon, Wilson C K

    2016-01-01

    The flagellated bacterium Escherichia coli is increasingly used experimentally as a self-propelled swimmer. To obtain meaningful, quantitative results that are comparable between different laboratories, reproducible protocols are needed to control, 'tune' and monitor the swimming behaviour of these motile cells. We critically review the knowledge needed to do so, explain methods for characterising the colloidal and motile properties of E. coli cells, and propose a protocol for keeping them swimming at constant speed at finite bulk concentrations. In the process of establishing this protocol, we use motility as a high-throughput probe of aspects of cellular physiology via the coupling between swimming speed and the proton motive force.

  3. Inducible repair of oxidative DNA damage in Escherichia coli.

    PubMed

    Demple, B; Halbrook, J

    Hydrogen peroxide is lethal to many cell types, including the bacterium Escherichia coli. Peroxides yield transient radical species that can damage DNA and cause mutations. Such partially reduced oxygen species are occasionally released during cellular respiration and are generated by lethal and mutagenic ionizing radiation. Because cells live in an environment where the threat of oxidative DNA damage is continual, cellular mechanisms may have evolved to avoid and repair this damage. Enzymes are known which evidently perform these functions. We report here that resistance to hydrogen peroxide toxicity can be induced in E. coli, that this novel induction is specific and occurs, in part, at the level of DNA repair.

  4. Sedimentation and gravitational instability of Escherichia coli Suspension

    NASA Astrophysics Data System (ADS)

    Douarche, Carine; Salin, Dominique; Collaboration between Laboratory FAST; LPS Collaboration

    2016-11-01

    The successive run and tumble of Escherichia coli bacteria provides an active matter suspension of rod-like particles with a large swimming diffusion. As opposed to inactive elongated particles, this diffusion prevents clustering and instability in the gravity field. We measure the time dependent E . coli concentration profile during their sedimentation. After some hours, due to the dioxygen consumption, a motile / non-motile front forms leading to a Rayleigh-Taylor type gravitational instability. Analyzing both sedimentation and instability in the framework of active particle suspensions, we can measure the relevant bacteria hydrodynamic characteristics such as its single particle sedimentation velocity and its hindrance volume.

  5. Advances in molecular serotyping and subtyping of Escherichia coli

    DOE PAGES

    Fratamico, Pina M.; DebRoy, Chitrita; Liu, Yanhong; ...

    2016-05-03

    Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtypingmore » and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsedfield gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. Furthermore, a variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.« less

  6. Gentamicin resistance among Escherichia coli strains isolated in neonatal sepsis.

    PubMed

    Hasvold, J; Bradford, L; Nelson, C; Harrison, C; Attar, M; Stillwell, T

    2013-01-01

    Neonatal sepsis is a significant cause of morbidity and mortality among term and preterm infants. Ampicillin and gentamicin are standard empiric therapy for early onset sepsis. Four cases of neonatal sepsis secondary to Escherichia coli (E. coli) found to be gentamicin resistant occurred within a five week period in one neonatal intensive care unit (NICU). To determine whether these cases could be tied to a single vector of transmission, and to more broadly evaluate the incidence of gentamicin resistant strains of E. coli in the neonatal population at our institution compared to other centers, we reviewed the charts of the four neonates (Infants A through D) and their mothers. The E. coli isolates were sent for Pulse Field Gel Electrophoresis (PFGE) to evaluate for genetic similarity between strains. We also reviewed all positive E. coli cultures from one NICU over a two year period. Infants A and B had genetically indistinguishable strains which matched that of urine and placental cultures of Infant B's mother. Infant C had a genetically distinct organism. Infant D, the identical twin of Infant C, did not have typing performed. Review of all cultures positive for E. coli at our institution showed a 12.9 percent incidence of gentamicin-resistance. A review of other studies showed that rates of resistance vary considerably by institution. We conclude that gentamicin-resistant E. coli is a relatively uncommon cause of neonatal sepsis, but should remain a consideration in patients who deteriorate despite initiation of empiric antibiotics.

  7. Inactivation of Escherichia coli using atmospheric-pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Kuwahata, Hiroshi; Yamaguchi, Takeshi; Ohyama, Ryu-ichiro; Ito, Atsushi

    2015-01-01

    An atmospheric-pressure argon (Ar) plasma jet was applied to the inactivation of Escherichia coli. The Ar plasma jet was generated at a frequency of 10 kHz, an applied voltage of 10 kV, and an Ar gas flow rate of 10 L/min at atmospheric pressure. E. coli cells seeded on an agar medium in a Petri dish were inactivated by Ar plasma jet irradiation for 1 s. Scanning electron microscopy (SEM) revealed that E. coli cells were killed because their cell wall and membrane were disrupted. To determine the causes of the disruption of the cell wall and membrane of E. coli, we performed the following experiments: the measurement of the surface temperature of an agar medium using a thermograph, the analysis of an emission spectrum of a plasma jet obtained using a multichannel spectrometer, and the determination of the distribution of the concentration of hydrogen peroxide (H2O2) generated on an agar medium by plasma jet irradiation using semiquantitative test strips. Moreover, H2O2 solutions of different concentrations were dropped onto an agar medium seeded with E. coli cells to examine the contribution of H2O2 to the death of E. coli. The results of these experiments showed that the cell wall and membrane of E. coli were disrupted by electrons in the plasma jet, as well as by electroneutral excited nitrogen molecules (N2) and hydroxyl (OH) radicals in the periphery of the plasma jet.

  8. Biosynthesis of phosphatidyl glycerophosphate in Escherichia coli.

    PubMed

    Chang, Y Y; Kennedy, E P

    1967-09-01

    An enzyme (L-glycerol 3-phosphate: CMP phosphatidyltransferase) catalyzing the synthesis of phosphatidyl glycerophosphate from CDP-diglyceride and L-glycerol 3-phosphate has been rendered soluble by treatment of the particulate, membrane-containing fraction of E. coli with Triton X-100 and has been partially purified. The enzyme, devoid of phosphatidyl glycerophosphatase activity, is specific for L-glycerol 3-phosphate and is completely dependent upon added Mg(++) or Mn(++) for activity. It has high affinity for CDP-diglyceride and can be used for the assay of this nucleotide. Other properties of the enzyme are also described.

  9. Growth and Division of Filamentous Forms of Escherichia coli.

    PubMed

    Adler, H I; Hardigree, A A

    1965-07-01

    Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Alice A. Hardigree. Growth and division of filamentous forms of Escherichia coli. J. Bacteriol. 90:223-226. 1965.-Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.

  10. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua.

    PubMed

    Tajkarimi, Mehrdad; Harrison, Scott H; Hung, Albert M; Graves, Joseph L

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.

  11. Enterotoxigenic Escherichia coli infection in captive black-footed ferrets.

    PubMed

    Bradley, G A; Orr, K; Reggiardo, C; Glock, R D

    2001-07-01

    Enterotoxigenic Escherichia coli with genes for heat stabile toxins Sta and STb was isolated from the gastrointestinal tract and multiple visceral organs of three adult and three juvenile black-footed ferrets (Mustela nigripes) that died in a captive breeding colony between 24 May 1998 and 2 July 1998. Similar isolates were obtained from rectal swabs of one adult and one juvenile that were clinically ill. All were fed a diet composed of mink chow, raw rabbit meat, beef liver powder, blood meal and lard. Escherichia coli of the same toxin genotype was isolated from the mixed ration. Clinical signs included sudden death, dehydration, anorexia and diarrhea. Necropsy lesions included acute enteritis with large numbers of rod shaped bacteria microscopically visible on intestinal villi.

  12. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

    PubMed Central

    Tajkarimi, Mehrdad; Harrison, Scott H.; Hung, Albert M.; Graves, Joseph L.

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism. PMID:26914334

  13. Thiolases of Escherichia coli: purification and chain length specificities.

    PubMed Central

    Feigenbaum, J; Schulz, H

    1975-01-01

    The presence of only one thiolase (EC 2.3.1.9) in wild-type Escherichia coli induced for enzymes of beta oxidation was demonstrated. A different thiolase was shown to be present in a mutant constitutive for the enzymes of butyrate degradation. The two thiolases were purified to near homogeneity by a simple two-step procedure and were found to be associated with different proteins as shown by gel electrophoresis. The thiolase isolated from induced wild-type Escherichia coli cell was active on beta-ketoacyl-coenzyme A derivatives containing 4 to 16 carbons, but exhibited optimal activity with medium-chain substrates. In contrast, the thiolase isolated from the constitutive mutant was shown to be specific for acetoacetyl-coenzyme A. PMID:236278

  14. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI III.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. III. Requirements for enzyme synthesis. J. Bacteriol. 84:996–1006. 1962.—The requirements for the formation of tryptophanase and tryptophan synthetase in Escherichia coli during repression release were studied. The kinetics of the formation of tryptophan synthetase differed in the two strains examined; this was attributed to differences in the endogenous level of tryptophan in the bacterial cells. The formation of both enzymes was inhibited by chloramphenicol, and by the absence of arginine in an arginine-requiring mutant. These results are indicative of a requirement for protein synthesis for enzyme formation. Requirements for nucleic acid synthesis were examined by use of a uracil- and thymine-requiring mutant, and with purine and pyrimidine analogues. The results obtained suggest that some type of ribonucleic acid synthesis was necessary for the formation of tryptophanase and tryptophan synthetase. PMID:13959620

  15. Penem derivatives: beta-lactamase stability and affinity for penicillin-binding proteins in Escherichia coli.

    PubMed

    Ohya, S; Utsui, Y; Sugawara, S; Yamazaki, M

    1982-03-01

    Penem derivatives, a new group of beta-lactam antibiotics with potent activities against a wide range of bacteria, including Pseudomonas aeruginosa, were tested for their stability against hydrolysis by beta-lactamases purified from clinical isolates of Morganella morganii. Proteus vulgaris, and Escherichia coli and by a penicillinase from Bacillus cereus. Penems having 6 alpha substituents, such as hydroxyethyl, hydroxymethyl, and ethyl groups, were very stable against hydrolysis by each of the enzymes. Penems having no 6 alpha substituents were easily hydrolyzed by P. vulgaris and E. coli enzymes, whereas they were rather stable against hydrolysis by M. morganii and B. cereus enzymes, a typical cephalosporinase and penicillinase, respectively. Affinity of the penems for E. coli penicillin-binding proteins (PBPs) was also tested. beta-Lactamase-stable penems having a 6 alpha-hydroxyethyl group showed high affinity for PBP-4, -5, and -6 as well as for PBP-1A, -1Bs, and -2. However, the penems having no 6 alpha substituents showed a far lower affinity for PBP-4, -5, and -6 than that shown by the corresponding 6 alpha-hydroxyethyl penems. Among the penems tested, affinity for PBP-4, -5, and -6 was closely related to their beta-lactamase stability, as was the case among cephamycins and cephalosporins. Effects of the penems on the morphology of a strain of E. coli are also described.

  16. Engineering of Escherichia coli to facilitate efficient utilization of isomaltose and panose in industrial glucose feedstock.

    PubMed

    Abe, Kenji; Kuroda, Akio; Takeshita, Ryo

    2017-03-01

    Industrial glucose feedstock prepared by enzymatic digestion of starch typically contains significant amounts of disaccharides such as maltose and isomaltose and trisaccharides such as maltotriose and panose. Maltose and maltosaccharides can be utilized in Escherichia coli fermentation using industrial glucose feedstock because there is an intrinsic assimilation pathway for these sugars. However, saccharides that contain α-1,6 bonds, such as isomaltose and panose, are still present after fermentation because there is no metabolic pathway for these sugars. To facilitate more efficient utilization of glucose feedstock, we introduced glvA, which encodes phospho-α-glucosidase, and glvC, which encodes a subunit of the phosphoenolpyruvate-dependent maltose phosphotransferase system (PTS) of Bacillus subtilis, into E. coli. The heterologous expression of glvA and glvC conferred upon the recombinant the ability to assimilate isomaltose and panose. The recombinant E. coli assimilated not only other disaccharides but also trisaccharides, including alcohol forms of these saccharides, such as isomaltitol. To the best of our knowledge, this is the first report to show the involvement of the microbial PTS in the assimilation of trisaccharides. Furthermore, we demonstrated that an L-lysine-producing E. coli harboring glvA and glvC converted isomaltose and panose to L-lysine efficiently. These findings are expected to be beneficial for industrial fermentation.

  17. Shear alters motility of Escherichia coli

    NASA Astrophysics Data System (ADS)

    Molaei, Mehdi; Jalali, Maryam; Sheng, Jian

    2013-11-01

    Understanding of locomotion of microorganisms in shear flows drew a wide range of interests in microbial related topics such as biological process including pathogenic infection and biophysical interactions like biofilm formation on engineering surfaces. We employed microfluidics and digital holography microscopy to study motility of E. coli in shear flows. We controlled the shear flow in three different shear rates: 0.28 s-1, 2.8 s-1, and 28 s-1 in a straight channel with the depth of 200 μm. Magnified holograms, recorded at 15 fps with a CCD camera over more than 20 minutes, are analyzed to obtain 3D swimming trajectories and subsequently used to extract shear responses of E.coli. Thousands of 3-D bacterial trajectories are tracked. The change of bacteria swimming characteristics including swimming velocity, reorientation, and dispersion coefficient are computed directly for individual trajectory and ensemble averaged over thousands of realizations. The results show that shear suppresses the bacterial dispersions in bulk but promote dispersions near the surface contrary to those in quiescent flow condition. Ongoing analyses are focusing to quantify effect of shear rates on tumbling frequency and reorientation of cell body, and its implication in locating the hydrodynamic mechanisms for shear enhanced angular scattering. NIH, NSF, GoMRI.

  18. Some factors affecting cyclopropane acid formation in Escherichia coli

    PubMed Central

    Knivett, V. A.; Cullen, Julia

    1965-01-01

    1. The fatty acid composition of the extractable lipids of Escherichia coli varied with growth conditions. 2. The principal fatty acids were palmitic acid, hexadecenoic acid, octadecenoic acid and the cyclopropane acids, methylenehexadecanoic acid and methyleneoctadecanoic acid. 3. Cyclopropane acid formation from monoenoic acids was increased by acid media, poor oxygen supply, or high growth temperature. 4. Cyclopropane acid formation was decreased by alkaline media, well oxygenated conditions, the presence of citrate, or lack of Mg2+. PMID:5324304

  19. Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli.

    PubMed

    Watanabe, Jun; Tanaka, Hisaki; Akagawa, Takumi; Mogi, Yoshinobu; Yamazaki, Tatsuo

    2007-10-01

    To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.

  20. Polymorphous crystallization and diffraction of threonine deaminase from Escherichia coli.

    PubMed

    Gallagher, D T; Eisenstein, E; Fisher, K E; Zondlo, J; Chinchilla, D; Yu, H D; Dill, J; Winborne, E; Ducote, K; Xiao, G; Gilliland, G L

    1998-05-01

    The biosynthetic threonine deaminase from Escherichia coli, an allosteric tetramer with key regulatory functions, has been crystallized in several crystal forms. Two distinct forms, both belonging to either space group P3121 or P3221, with different sized asymmetric units that both contain a tetramer, grow under identical conditions. Diffraction data sets to 2.8 A resolution (native) and 2. 9 A resolution (isomorphous uranyl derivative) have been collected from a third crystal form in space group I222.

  1. Positive regulation of the Escherichia coli glycine cleavage enzyme system.

    PubMed Central

    Wilson, R L; Steiert, P S; Stauffer, G V

    1993-01-01

    A new mutation in Escherichia coli, designated gcvA1, that results in noninducible expression of both gcv and a gcvT-lacZ gene fusion was isolated. A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression. These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv. PMID:8423160

  2. Division pattern of a round mutant of Escherichia coli.

    PubMed Central

    Cooper, S

    1997-01-01

    A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads. This implies that successive division planes of the round mutant are parallel rather than perpendicular. These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W. D. Donachie, S. Addinall, and K. Begg, Bioessays 17:569-576, 1995). PMID:9287016

  3. Antibacterial efficacy of silver nanoparticles against Escherichia coli

    NASA Astrophysics Data System (ADS)

    Pattabi, Rani M.; Thilipan, G. Arun Kumar; Bhat, Vinayachandra; Sridhar, K. R.; Pattabi, Manjunatha

    2013-02-01

    Silver nanoparticles (AgNPs) synthesized by subjecting an aqueous solution of AgNO3 and polyvinyl alcohol to irradiation from an UV lamp has been studied for its antibacterial potential against Gram-negative bacteria (Escherichia coli). The diameter of the zone of inhibition is found to depend on both the irradiation time and the nanoparticle concentration. As the synthesis method adopted uses no toxic reagents, these particles may serve as promising candidates in the search for better antibacterial agents.

  4. Electric field induced bacterial flocculation of Enteroaggregative Escherichia coli 042

    SciTech Connect

    Kumar, Aloke; Mortensen, Ninell P; Mukherjee, Partha P; Retterer, Scott T; Doktycz, Mitchel John

    2011-01-01

    A response of the aggregation dynamics of enteroaggregative Escherichia coli under low magnitude steady and oscillating electric fields is presented. The presence of uniform electric fields hampered microbial adhesion and biofilm formation on a transverse glass surface, but instead promoted the formation of flocs. Extremely heterogeneous distribution of live and dead cells was observed among the flocs. Moreover, floc formation was largely observed to be independent of the frequency of alternating electric fields.

  5. Role for the female in bacterial conjugation in Escherichia coli.

    PubMed

    Freifelder, D

    1967-08-01

    Hfr and F' Lac male strains of Escherichia coli were mated with purine-requiring females which had been starved for purine. These females formed mating pairs with the males. However, a mating in the absence of purine markedly reduced the yield of recombinants. Transfer of F' Lac or of lambda prophage also occurred infrequently. It was concluded that deoxyribonucleic acid transfer from male to female requires some, as yet unknown, function of the female.

  6. Role for the Female in Bacterial Conjugation in Escherichia coli

    PubMed Central

    Freifelder, David

    1967-01-01

    Hfr and F′ Lac male strains of Escherichia coli were mated with purine-requiring females which had been starved for purine. These females formed mating pairs with the males. However, a mating in the absence of purine markedly reduced the yield of recombinants. Transfer of F′ Lac or of λ prophage also occurred infrequently. It was concluded that deoxyribonucleic acid transfer from male to female requires some, as yet unknown, function of the female. PMID:5341864

  7. Current perspectivesin pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli.

    PubMed

    Kong, Haishen; Hong, Xiaoping; Li, Xuefen

    2015-08-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen that causes acute and persistent diarrhea in children and adults. While the pathogenic mechanisms of EAEC intestinal colonization have been uncovered (including bacterial adhesion, enterotoxin and cytotoxin secretion, and stimulation of mucosal inflammation), those of severe extraintestinal infections remain largely unknown. The recent emergence of multidrug resistant EAEC represents an alarming public health threat and clinical challenge, and research on the molecular mechanisms of resistance is urgently needed.

  8. Lipophilic chelator inhibition of electron transport in Escherichia coli.

    PubMed Central

    Crane, R T; Sun, I L; Crane, F L

    1975-01-01

    The lipophilic chelator bathophenanthroline inhibits electron transport in membranes from Escherichia coli. The less lipophilic 1,10-phenanthroline, bathophenanthroline sulfonate, and alpha,alpha-dipyridyl have little effect. Reduced nicotinamide adenine dinucleotide oxidase is more sensitive to bathophenanthroline inhibition than lactate oxidase activity. Evidence for two sites of inhibition comes from the fact that both reduced nicotinamide adenine dinucleotide menadione reductase and duroquinol oxidase activities are inhibited. Addition of uncouplers of phosphorylation before bathophenanthroline protects against inhibition. PMID:1092663

  9. Effects of Acridine Orange on the Growth of Escherichia coli

    PubMed Central

    Southwick, Frederick S.; Carr, Howard S.; Carden, George A.; D'Alisa, Rose M.; Rosenkranz, Herbert S.

    1972-01-01

    Exposure of Escherichia coli to critical acridine orange (AO) concentrations did not result in loss of viability. However, the deoxyribonucleic acid (DNA) of cells exposed to such agents was rapidly degraded and repolymerized. On the other hand, a bacterium deficient in DNA repair (pol A1−, lacking DNA polymerase) was sensitive to the action of AO. The DNA of such cells was also degraded but it was not repaired. PMID:4553001

  10. Two Forms of d-Glycerate Kinase in Escherichia coli

    PubMed Central

    Ornston, M. K.; Ornston, L. N.

    1969-01-01

    Escherichia coli K-12 synthesizes two chromatographically distinct forms of glycerate kinase which differ both in their thermolability and in the dependence of their activity upon pH. One enzymatic form, GK I, is found in cells grown with glycerate, glucarate, or glycolate. Of these compounds, glycolate is the only carbon source that elicits the synthesis of the second enzymatic form, GK II. PMID:4887503

  11. Preparation of Soluble Proteins from Escherichia coli

    PubMed Central

    Wingfield, Paul T.

    2014-01-01

    Purification of human IL-1β is used in this unit as an example of the preparation of soluble proteins from E. coli. Bacteria containing IL-1β are lysed, and IL-1 β in the resulting supernatant is purified by anion-exchange chromatography, salt precipitation and cation-exchange chromatography, and then concentrated. Finally, the IL-1 β protein is applied to a gel-filtration column to separate it from remaining higher- and lower-molecular-weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state. Also, the purification procedure serves as an example of how use classical protein purifications methods which may also be used in conjunction with the affinity-based methods now more commonly used. PMID:25367009

  12. Fluorogenic assays for immediate confirmation of Escherichia coli.

    PubMed Central

    Feng, P C; Hartman, P A

    1982-01-01

    Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains). Images PMID:7049088

  13. Prevalence of Escherichia coli in apple cider manufactured in Connecticut.

    PubMed

    Dingman, D W

    1999-06-01

    Cider samples obtained from 11 cider mills operating in Connecticut during the 1997 to 1998 production season were tested for the presence of Escherichia coli. Cider production began in mid August and continued through March, with peak production in September and October. Of 314 cider samples tested, 11 (4%) were found to contain E. coli. Of the 11 mills, 6 (55%) tested positive for E. coli in the cider at least once during the production year. E. coli was first observed in cider samples produced in mid to late October and was not detected in samples made after January. A trend was observed for cider to decrease in acidity and increase in Brix (soluble sugars) throughout the production season. No correlation between pH and soluble sugars of cider and the presence of E. coli was detected. Eight mills used both dropped apples and tree-picked apples, whereas three mills used tree-picked apples only. The use of dropped apples in cider production began 5 weeks before the first detection of E. coli in cider. E. coli was isolated from cider samples produced using dropped apples and from samples produced using only tree-picked apples. No direct correlation between the use of dropped apples or tree-picked apples and the presence of E. coli in the cider was observed. An association between the time of apple harvest and the appearance of E. coli in cider was noted. For mills providing adequate records, all contaminated cider was produced from apples harvested between mid October and mid November.

  14. Thymineless Death in Escherichia coli: Inactivation and Recovery

    PubMed Central

    Cummings, Donald J.; Kusy, Alvin R.

    1969-01-01

    The effects of chloramphenicol (CAP) on the progress of thymineless death (TLD), nalidixic acid (NA) inactivation, ultraviolet (UV) irradiation, and mitomycin C (MC) inactivation were studied in Escherichia coli B, Bs−1, Bs−3, Bs−12, and B/r. This was done before, during, and after inactivation. During the progress of inactivation, it was found that at 10 to 20 μg of CAP per ml, up to 50% of the UV-sensitive bacteria survived TLD and about 10% survived NA. In E. coli B/r, at these concentrations of CAP, about 10 to 15% of the cells survived TLD and about 20 to 25% survived NA. Concentrations of CAP greater than 25 μg/ml actually increased the sensitivity of E. coli B, Bs−1, Bs−3, and Bs−12 to inactivation by either TLD or NA; at 150 μg of CAP per ml, the sensitivity of E. coli B/r to inactivation also increased. When E. coli B cells were incubated in CAP prior to inactivation, the longer the preincubation the longer onset of TLD was delayed; NA inactivation was also affected in that the rate of inactivation after CAP incubation was greatly decreased. Preincubation of E. coli B/r with CAP had much less effect on the progress of inactivation. After thymineless death, incubation in CAP plus thymine led to a rapid and almost complete recovery of E. coli B and Bs−12. Lesser recoveries were observed after inactivation due to UV, NA, or MC inactivation. E. coli Bs−1 and B/r did not recover viability after any mode of inactivation, and E. coli Bs−3 and Bs−12 recovered from UV to about 20% of the initial titer. It was suggested that protein synthesis, in particular proteins involved in deoxyribonucleic synthesis, was a determining factor in these inactivating and recovery events. PMID:4897115

  15. Cajanol inhibits the growth of Escherichia coli and Staphylococcus aureus by acting on membrane and DNA damage.

    PubMed

    Liu, Xiao-lei; Zhang, Xin-jian; Fu, Yu-jie; Zu, Yuan-gang; Wu, Nan; Liang, Lu; Efferth, Thomas

    2011-01-01

    In the present study, the mechanism of antibacterial activity of cajanol extracted from the roots of Cajanus cajan (L.) Millsp. towards Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated. The antibacterial activity of cajanol was evaluated towards six bacterial strains (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa) by the broth microdilution method. It showed strong antibacterial activity towards all bacteria tested with minimal inhibition concentration (MIC) values ranging from 98.90 µM to 197.8 µM. Cajanol-induced death rates in the most sensitive strains ( E.COLI, 96.55 % and S. AUREUS, 97.25 %) were analyzed by flow cytometry. Furthermore, the activity of cajanol on the membranes of E. coli and S. aureus was investigated by using lecithin, phosphate groups, and fluorescence microscopy. Cajanol-induced DNA damage was observed by agarose gel electrophoresis. In summary, cajanol inhibited E. coli only by DNA damage, whereas S. aureus was inhibited by affecting both, the lecithin and phosphate groups on the cellular membrane and DNA. The present study shows that cajanol possesses antibacterial activity in vitro towards both gram-negative and gram-positive bacteria and therefore may be a promising candidate as an antibacterial agent for the therapy of microbial infections.

  16. Interaction of Escherichia coli and Soil Particles in Runoff

    PubMed Central

    Muirhead, Richard William; Collins, Robert Peter; Bremer, Philip James

    2006-01-01

    A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland flow were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (>45 μm). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (<2 μm) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix. PMID:16672484

  17. Measuring Escherichia coli Gene Expression during Human Urinary Tract Infections

    PubMed Central

    Mobley, Harry L. T.

    2016-01-01

    Extraintestinal Escherichia coli (E. coli) evolved by acquisition of pathogenicity islands, phage, plasmids, and DNA segments by horizontal gene transfer. Strains are heterogeneous but virulent uropathogenic isolates more often have specific fimbriae, toxins, and iron receptors than commensal strains. One may ask whether it is the virulence factors alone that are required to establish infection. While these virulence factors clearly contribute strongly to pathogenesis, bacteria must survive by metabolizing nutrients available to them. By constructing mutants in all major metabolic pathways and co-challenging mice transurethrally with each mutant and the wild type strain, we identified which major metabolic pathways are required to infect the urinary tract. We must also ask what else is E. coli doing in vivo? To answer this question, we examined the transcriptome of E. coli CFT073 in the murine model of urinary tract infection (UTI) as well as for E. coli strains collected and analyzed directly from the urine of patients attending either a urology clinic or a university health clinic for symptoms of UTI. Using microarrays and RNA-seq, we measured in vivo gene expression for these uropathogenic E. coli strains, identifying genes upregulated during murine and human UTI. Our findings allow us to propose a new definition of bacterial virulence. PMID:26784237

  18. Regulation of arabinose and xylose metabolism in Escherichia coli.

    PubMed

    Desai, Tasha A; Rao, Christopher V

    2010-03-01

    Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.

  19. Escherichia coli sequence type 131: epidemiology and challenges in treatment.

    PubMed

    Qureshi, Zubair A; Doi, Yohei

    2014-05-01

    Escherichia coli ST131 has emerged as a global epidemic, multidrug-resistant clone of E. coli causing extra-intestinal infections. It is now highly prevalent among fluoroquinolone-resistant and CTX-M ESBL-producing E. coli isolates worldwide. Humans are likely the primary reservoir of ST131. Factors associated with its acquisition include residence in long-term care facilities and recent receipt of antimicrobial agents. E. coli ST131 causes a wide array of infections ranging from cystitis to life-threatening sepsis. Fluoroquinolones and trimethoprim-sulfamethoxazole are no longer adequate options for empiric therapy when E. coli ST131 is suspected from risk factors and local epidemiology. Expanded-spectrum cephalosporins, piperacillin-tazobactam and carbapenems are options to treat serious non-ESBL-producing E. coli ST131 infections, while carbapenems are indicated for ESBL-producing infections. There is a growing interest in reevaluating oral agents including fosfomycin and pivmecillinam for less serious infections such as uncomplicated cystitis.

  20. Characterization of Shiga toxigenic Escherichia coli isolated from foods.

    PubMed

    Martínez, Aida Juliana; Bossio, Carolina Paba; Durango, Adriana Coral; Vanegas, Maria Consuelo

    2007-12-01

    The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.

  1. [Escherichia coli, a pathogen under fire from the news].

    PubMed

    Cohen, R; Raymond, J; Gendrel, D; Bingen, E

    2012-11-01

    Escherichia coli is both a gastrointestinal tract commensal and a major pathogen. In recent years, E. coli is under fire from the news due to a better understanding of pathogenic factors, outbreaks of infections caused by enterohaemorrhagic strains, and last but not least, the worrying development of antibiotic resistance. Due to the absence of new compounds active against these strains, producing extended-spectrum ß-lactamases (ESBL) and frequently multiresistant to other antibiotics, their emergence will pose therapeutic problems for practitioners of all pediatric specialties. The gold standard treatment for severe infections due to ESBL-E. coli family is the penem class. The frequent use of penems promotes the emergence of strains resistant to carbapenems. Sparing carbapenems should be a clear objective for non life-threatening infections.

  2. Functions of the gene products of Escherichia coli.

    PubMed Central

    Riley, M

    1993-01-01

    A list of currently identified gene products of Escherichia coli is given, together with a bibliography that provides pointers to the literature on each gene product. A scheme to categorize cellular functions is used to classify the gene products of E. coli so far identified. A count shows that the numbers of genes concerned with small-molecule metabolism are on the same order as the numbers concerned with macromolecule biosynthesis and degradation. One large category is the category of tRNAs and their synthetases. Another is the category of transport elements. The categories of cell structure and cellular processes other than metabolism are smaller. Other subjects discussed are the occurrence in the E. coli genome of redundant pairs and groups of genes of identical or closely similar function, as well as variation in the degree of density of genetic information in different parts of the genome. PMID:7508076

  3. Incidence of Escherichia coli in Black Walnut Meats

    PubMed Central

    Meyer, Melvin T.; Vaughn, Reese H.

    1969-01-01

    Examination of commercially shelled black walnut meats showed inconsistent numbers of total aerobic bacteria, coliforms, and Escherichia coli; variation occurred among different meat sizes and within each meat size. The incidence of E. coli on meats of commercially hulled black walnuts depended on the physical condition of the nuts. Apparently tightly sealed ones contained only a few or none, whereas those with visibly separated sutures and spoiled meats yielded the most. This contamination was in part correlated to a hulling operation. Large numbers of E. coli on the husk of the walnuts contaminated the hulling water, subsequently also contaminating the meats by way of separated sutures. Chlorination of the hulling wash water was ineffective. Attempts were made to decontaminate the walnut meats without subsequent deleterious changes in flavor or texture. A treatment in coconut oil at 100 C followed by removal of excess surface oil by centrifugation was best. PMID:4905608

  4. Quantitative method for enumeration of enterotoxigenic Escherichia coli.

    PubMed Central

    Calderon, R L; Levin, M A

    1981-01-01

    A rapid method was developed to quantify toxigenic Escherichia coli, using a membrane filter procedure. After filtration of samples, the membrane filter was first incubated on a medium selective for E. coli (24 h, 44 degrees C) and then transferred to tryptic soy agar (3%; 6 h, 37 degrees C). To assay for labile toxin-producing colonies, the filter was then transferred to a monolayer of Y-1 cells, the E. coli colonies were marked on the bottom of the petri dish, and the filter was removed after 15 min. The monolayer was observed for a positive rounding effect after a 15- to 24-h incubation. The method has an upper limit of detecting 30 toxigenic colonies per plate and can detect as few as one toxigenic colony per plate. A preliminary screening for these enterotoxigenic strains in polluted waters and known positive fecal samples was performed, and positive results were obtained with fecal samples only. PMID:7007415

  5. Mechanisms of the radioprotective effect of cysteamine in Escherichia coli

    SciTech Connect

    Korystov, Yu.N.; Vexler, F.B.

    1988-06-01

    The values of the oxygen effect (m) and the maximal protective effect of cysteamine (DMF*) were estimated for four Escherichia coli strains: AB1157 (wild type), AB1886 (uvrA), AB2463 (recA), and p3478 (polA). A correlation made between DMF* and m as well as the kinetics of the increase of DMF with oxygen depletion showed that the protective effect of cysteamine is realized by three mechanisms: (i) anoxia achieved by oxygen reduction, with the DMF varying from 2.2 to 4.2 for different E. coli strains (this protection is the major contribution to the entire mechanism); (ii) lowering of the indirect radiation effect; i.e., for 50 mM cysteamine DMF does not exceed 1.1; and (iii) increase of the efficiency of enzymatic repair. The latter effect of cysteamine is registered only with the wild-type E. coli, the DMF being not less than 1.4.

  6. Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli

    PubMed Central

    Laganenka, Leanid; Colin, Remy; Sourjik, Victor

    2016-01-01

    Bacteria communicate by producing and sensing extracellular signal molecules called autoinducers. Such intercellular signalling, known as quorum sensing, allows bacteria to coordinate and synchronize behavioural responses at high cell densities. Autoinducer 2 (AI-2) is the only known quorum-sensing molecule produced by Escherichia coli but its physiological role remains elusive, although it is known to regulate biofilm formation and virulence in other bacterial species. Here we show that chemotaxis towards self-produced AI-2 can mediate collective behaviour—autoaggregation—of E. coli. Autoaggregation requires motility and is strongly enhanced by chemotaxis to AI-2 at physiological cell densities. These effects are observed regardless whether cell–cell interactions under particular growth conditions are mediated by the major E. coli adhesin (antigen 43) or by curli fibres. Furthermore, AI-2-dependent autoaggregation enhances bacterial stress resistance and promotes biofilm formation. PMID:27687245

  7. Engineering Escherichia coli K12 MG1655 to use starch

    PubMed Central

    2014-01-01

    Background To attain a sustainable bioeconomy, fuel, or valuable product, production must use biomass as substrate. Starch is one of the most abundant biomass resources and is present as waste or as a food and agroindustry by-product. Unfortunately, Escherichia coli, one of the most widely used microorganisms in biotechnological processes, cannot use starch as a carbon source. Results We engineered an E. coli strain capable of using starch as a substrate. The genetic design employed the native capability of the bacterium to use maltodextrins as a carbon source plus expression and secretion of its endogenous α-amylase, AmyA, in an adapted background. Biomass production improved using 35% dissolved oxygen and pH 7.2 in a controlled bioreactor. Conclusion The engineered E. coli strain can use starch from the milieu and open the possibility of optimize the process to use agroindustrial wastes to produce biofuels and other valuable chemicals. PMID:24886307

  8. Gentamicin-loaded borate bioactive glass eradicates osteomyelitis due to Escherichia coli in a rabbit model.

    PubMed

    Xie, Zongping; Cui, Xu; Zhao, Cunju; Huang, Wenhai; Wang, Jianqiang; Zhang, Changqing

    2013-07-01

    The treatment of osteomyelitis induced by Gram-negative bacilli is rarely reported in the literature. This study established a rabbit tibia model of osteomyelitis induced by the Gram-negative bacillus Escherichia coli. Using this model, pellets composed of a chitosan-bonded mixture of borate bioactive glass and gentamicin were evaluated in vitro and in vivo for the treatment of osteomyelitis induced by Escherichia coli. Our results showed that the pellets in phosphate-buffered saline released gentamicin continuously over 26 days. Without the simultaneous use of a systemic antibiotic, the implantation of the gentamicin-loaded pellets into the osteomyelitis region of the tibia resulted in the eradication of 81.82% of infections, as determined by microbiological, histological and radiographic evaluation, and supported the ingrowth of new bone into the tibia defects after 6 weeks of implantation. The results indicate that the gentamicin-loaded borate bioactive glass implant, combining sustained drug release with the ability to support new bone formation, could provide a method for treating osteomyelitis induced by Gram-negative bacilli.

  9. Gentamicin-Loaded Borate Bioactive Glass Eradicates Osteomyelitis Due to Escherichia coli in a Rabbit Model

    PubMed Central

    Xie, Zongping; Cui, Xu; Zhao, Cunju; Huang, Wenhai; Wang, Jianqiang

    2013-01-01

    The treatment of osteomyelitis induced by Gram-negative bacilli is rarely reported in the literature. This study established a rabbit tibia model of osteomyelitis induced by the Gram-negative bacillus Escherichia coli. Using this model, pellets composed of a chitosan-bonded mixture of borate bioactive glass and gentamicin were evaluated in vitro and in vivo for the treatment of osteomyelitis induced by Escherichia coli. Our results showed that the pellets in phosphate-buffered saline released gentamicin continuously over 26 days. Without the simultaneous use of a systemic antibiotic, the implantation of the gentamicin-loaded pellets into the osteomyelitis region of the tibia resulted in the eradication of 81.82% of infections, as determined by microbiological, histological and radiographic evaluation, and supported the ingrowth of new bone into the tibia defects after 6 weeks of implantation. The results indicate that the gentamicin-loaded borate bioactive glass implant, combining sustained drug release with the ability to support new bone formation, could provide a method for treating osteomyelitis induced by Gram-negative bacilli. PMID:23629702

  10. Enhanced secreting expression and improved properties of a recombinant alkaline endoglucanase cloned in Escherichia coli.

    PubMed

    Liu, Sen-Lin; Chen, Wei-Zhao; Liu, Gang; Xing, Miao

    2011-07-01

    An alkaline endoglucanase from Bacillus akibai III-3A was successfully expressed in Escherichia coli in active form, and secretion was greatly enhanced by addition of 5 g/l ethylenediamine tetraacetic acid (EDTA) to the culture medium at the induction time of 12 h. Under the optimal culture conditions, extracellular and total endoglucanase activities were 18.5 and 31.2 U/ml, respectively. Both the recombinant and native enzymes exhibited similar properties with respect to broad pH stability, good thermostability, and resistibility to various metal ions and reagents examined. However, unlike the native endoglucanase that was partly inhibited by sodium dodecyl sulfate (SDS), the recombinant enzyme had good resistibility to SDS, being very stable in the commercial detergents, and no decrease in residual activity was observed in 0.2% (w/v) laundry detergent, indicating that it was suitable for application in detergents industry.

  11. Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli

    SciTech Connect

    Young, C.C.; Alvarez, J.D.; Bernlohr, R.W. )

    1990-09-01

    Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and (methyl-3H)methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis and are consistent with the proposal that methylation of this protein functions in nutrient sensing.

  12. Crystal structure of Escherichia coli YidC, a membrane protein chaperone and insertase.

    PubMed

    Kumazaki, Kaoru; Kishimoto, Toshiki; Furukawa, Arata; Mori, Hiroyuki; Tanaka, Yoshiki; Dohmae, Naoshi; Ishitani, Ryuichiro; Tsukazaki, Tomoya; Nureki, Osamu

    2014-12-03

    Bacterial YidC, an evolutionally conserved membrane protein, functions as a membrane protein chaperone in cooperation with the Sec translocon and as an independent insertase for membrane proteins. In Gram-negative bacteria, the transmembrane and periplasmic regions of YidC interact with the Sec proteins, forming a multi-protein complex for Sec-dependent membrane protein integration. Here, we report the crystal structure of full-length Escherichia coli YidC. The structure reveals that a hydrophilic groove, formed by five transmembrane helices, is a conserved structural feature of YidC, as compared to the previous YidC structure from Bacillus halodurans, which lacks a periplasmic domain. Structural mapping of the substrate- or Sec protein-contact sites suggested the importance of the groove for the YidC functions as a chaperone and an insertase, and provided structural insight into the multi-protein complex.

  13. Expression of thermostable β-xylosidase in Escherichia coli for use in saccharification of plant biomass.

    PubMed

    Aftab, Muhammad N; Zafar, Asma; Awan, Ali R

    2017-01-31

    The present work is aimed to evaluate the saccharification potential of a thermostable β-xylosidase cloned from Bacillus licheniformis into Escherichia coli for production of bioethanol from plant biomass. Recombinant β-xylosidase enzyme possesses the ability of bioconversion of plant biomass like wheat straw, rice straw and sugarcane bagass. By using this approach, plant biomass that mainly constitute cellulose can be converted to reducing sugars that could then be easily converted to bioethanol by simple fermentation process. The production of bioethanol will help to overcome energy requirements due to depleting fossil fuels and will also help to protect environment by reducing greenhouse gas emission. In the end, future directions are briefly mentioned that can be utilized to reduce the cost and increase the yield of biofuels.

  14. Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway.

    PubMed

    Zhao, Yaru; Yang, Jianming; Qin, Bo; Li, Yonghao; Sun, Yuanzhang; Su, Sizheng; Xian, Mo

    2011-06-01

    Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-D: -xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.

  15. Effect of tannins on the in viro growth of Escherichia coli O157:H7 and in vivo growth of generic Escherichia coli excreted from steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement o...

  16. Effective medicinal plants against enterohaemorrhagic Escherichia coli O157:H7.

    PubMed

    Voravuthikunchai, Supayang; Lortheeranuwat, Amornrat; Jeeju, Wanpen; Sririrak, Trechada; Phongpaichit, Souwalak; Supawita, Thanomjit

    2004-09-01

    The stimulating effect of subinhibitory concentrations of antibiotics on the production of verocytotoxin (VT) by enterohaemorrhagic Escherichia coli (EHEC) O157:H7 has been claimed. The purpose of this study was to find an alternative, but bioactive medicine for the treatment of this organism. Fifty-eight preparations of aqueous and ethanolic extracts of 38 medicinal plant species commonly used in Thailand to cure gastrointestinal infections were tested for their antibacterial activity against different strains of Escherichia coli, including 6 strains of Escherichia coli O157:H7, Escherichia coli O26:H11, Escherichia coli O111:NM, Escherichia coli O22; 5 strains of Escherichia coli isolated from bovine; and Escherichia coli ATCC 25922. Inhibition of growth was primarily tested by the paper disc agar diffusion method. Among the medicinal plants tested, only 8 species (21.05%) exhibited antimicrobial activity against Escherichia coli O157:H7. Acacia catechu, Holarrhena antidysenterica, Peltophorum pterocarpum, Psidium guajava, Punica granatum, Quercus infectoria, Uncaria gambir, and Walsura robusta demonstrated antibacterial activity with inhibition zones ranging from 7 to 17 mm. The greatest inhibition zone against Escherichia coli O157:H7 (RIMD 05091083) was produced from the ethanolic extract of Quercus infectoria. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the agar microdilution method and agar dilution method in petri dishes with millipore filter. Both aqueous and ethanolic extracts of Quercus infectoria and aqueous extract of Punica granatum were highly effective against Escherichia coli O157:H7 with the best MIC and MBC values of 0.09, 0.78, and 0.19, 0.39 mg/ml, respectively. These plant species may provide alternative but bioactive medicines for the treatment of Escherichia coli O157:H7 infection.

  17. Soil solarization reduces Escherichia coli O157:H7 and total Escherichia coli on cattle feedlot pen surfaces.

    PubMed

    Berry, Elaine D; Wells, James E

    2012-01-01

    Feedlot pen soil is a source for transmission of Escherichia coli O157:H7, and therefore a target for preharvest strategies to reduce this pathogen in cattle. The objective of this study was to determine the ability of soil solarization to reduce E. coli O157:H7 in feedlot surface material (FSM). A feedlot pen was identified in which naturally occurring E. coli O157:H7 was prevalent and evenly distributed in the FSM. Forty plots 3 by 3 m were randomly assigned such that five plots of each of the solarization times of 0, 1, 2, 3, 4, 6, 8, and 10 weeks were examined. Temperature loggers were placed 7.5 cm below the surface of each plot, and plots to be solarized were covered with clear 6-mil polyethylene. At each sampling time, five FSM samples were collected from each of five solarized and five unsolarized plots. E. coli concentrations and E. coli O157:H7 presence by immunomagnetic separation and plating were determined for each FSM sample. Initial percentages of E. coli O157:H7-positive samples in control and solarized FSM were 84 and 80%, respectively, and did not differ (P > 0.05). E. coli O157:H7 was no longer detectable by 8 weeks of solarization, but was still detected in unsolarized FSM at 10 weeks. The average initial concentration of E. coli in FSM was 5.56 log CFU/g and did not differ between treatments (P > 0.05). There was a 2.0-log decrease of E. coli after 1 week of solarization, and a >3.0-log reduction of E. coli by week 6 of solarization (P, 0.05). E. coli levels remained unchanged in unsolarized FSM (P > 0.05). Daily peak FSM temperatures were on average 8.7°C higher for solarized FSM compared with unsolarized FSM, and reached temperatures as high as 57°C. Because soil solarization reduces E. coli O157:H7, this technique may be useful for reduction of persistence and transmission of this pathogen in cattle production, in addition to remediation of E. coli O157:H7-contaminated soil used to grow food crops.

  18. Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner.

    PubMed

    Yu, Huili; Li, Haoran; Gao, Dongfang; Gao, Cuijuan; Qi, Qingsheng

    2015-11-20

    Antimicrobial peptides (AMPs) are small molecules which serve as essential components of the innate immune system in various organisms. AMPs possess a broad spectrum of antimicrobial activities. However, the scaled production of such peptides in Escherichia coli faces many difficulties because of their small size and toxicity to the host. Here, we described a new fusion strategy to extracellularly produce significant amounts of these antimicrobial peptides in recombinant E. coli at significant amount. Employing the catalytic domain of a cellulase (Cel-CD) from Bacillus subtilis KSM-64 as the fusion partner, five recombinant antimicrobial peptides were confirmed to accumulate in the culture medium at concentrations ranging from 184 mg/L to 297 mg/L. The radical diffusion experiment demonstrated that the released model antimicrobial peptide, bombinin, had antibacterial activities against both E. coli and Staphylococcus aureus. This strategy will be suitable for the production of antimicrobial peptides and other toxicity proteins.

  19. 77 FR 26725 - Changes to FSIS Traceback, Recall Procedures for Escherichia coli O157:H7 Positive Raw Beef...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-07

    ... Food Safety and Inspection Service Changes to FSIS Traceback, Recall Procedures for Escherichia coli... find raw ground beef presumptive positive for Escherichia coli (E. coli) O157:H7. This methodology will... Escherichia coli O157:H7'' and requested comments on these documents. FSIS also held a public meeting...

  20. Antibiotic Resistance in Urinary Isolates of Escherichia coli

    PubMed Central

    Abduzaimovic, Amila; Aljicevic, Mufida; Rebic, Velma; Vranic, Sabina Mahmutovic; Abduzaimovic, Kadrija; Sestic, Sabina

    2016-01-01

    Objectives: The aim of this study was to examine the presence of antimicrobial resistance / susceptibility strains of Escherichia coli in inpatients and outpatients. Materials and methods: It is a retrospective study carried out at the Department of Microbiology, Parasitology and Virology Faculty of Medicine, University of Sarajevo. In cooperation with the Microbiological laboratory of the Cantonal Hospital Zenica and the Microbiological laboratory of the General Hospital Tesanj, 3863 urine samples were processed in the period from March 1st to March 31st 2016. Results: Our study showed that E. coli had the highest antimicrobial resistance to trimethoprim / sulfamethoxazole (38.61%), followed by amoxicillin / clavulanic acid (19.62%), ciprofloxacin (9.49%), gentamicin (8.86%), cephalexin (8.23%), nitrofurantoin (8.23%), cefuroxime (7.52%), ceftazidime (6.33%), cefuroxime (89.87%), amikacin (4.43%). Conclusions: The isolated strains of E. coli showed the highest resistance to trimethoprim / sulfamethoxazole and amoxicillin / clavulanic acid. The isolated strains of E. coli showed the greatest susceptibility to amikacin and ceftazidime. Gender distribution of positive E. coli isolates showed statistically significant differences in favor of females. PMID:28144190

  1. Antibiotic Resistance of Escherichia coli Serotypes from Cochin Estuary

    PubMed Central

    Sukumaran, Divya P.; Durairaj, Srinivasan; Abdulla, Mohamed Hatha

    2012-01-01

    This study aimed at detecting the prevalence of antibiotic-resistant serotypes of Escherichia coli in Cochin estuary, India. E. coli strains were isolated during the period January 2010–December 2011 from five different stations set at Cochin estuary. Water samples from five different stations in Cochin estuary were collected on a monthly basis for a period of two years. Isolates were serotyped, antibiogram-phenotyped for twelve antimicrobial agents, and genotyped by polymerase chain reaction for uid gene that codes for β-D-glucuronidase. These E. coli strains from Cochin estuary were tested against twelve antibiotics to determine the prevalence of multiple antibiotic resistance among them. The results revealed that more than 53.33% of the isolates were multiple antibiotic resistant. Thirteen isolates showed resistance to sulphonamides and two of them contained the sul 1 gene. Class 1 integrons were detected in two E. coli strains which were resistant to more than seven antibiotics. In the present study, O serotyping, antibiotic sensitivity, and polymerase chain reaction were employed with the purpose of establishing the present distribution of multiple antibiotic-resistant serotypes, associated with E. coli isolated from different parts of Cochin estuary. PMID:23008708

  2. Escherichia coli ST131, an Intriguing Clonal Group

    PubMed Central

    Bertrand, Xavier; Madec, Jean-Yves

    2014-01-01

    SUMMARY In 2008, a previously unknown Escherichia coli clonal group, sequence type 131 (ST131), was identified on three continents. Today, ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide. Retrospective studies have suggested that it may originally have risen to prominence as early as 2003. Unlike other classical group B2 ExPEC isolates, ST131 isolates are commonly reported to produce extended-spectrum β-lactamases, such as CTX-M-15, and almost all are resistant to fluoroquinolones. Moreover, ST131 E. coli isolates are considered to be truly pathogenic, due to the spectrum of infections they cause in both community and hospital settings and the large number of virulence-associated genes they contain. ST131 isolates therefore seem to contradict the widely held view that high levels of antimicrobial resistance are necessarily associated with a fitness cost leading to a decrease in pathogenesis. Six years after the first description of E. coli ST131, this review outlines the principal traits of ST131 clonal group isolates, based on the growing body of published data, and highlights what is currently known and what we need to find out to provide public health authorities with better information to help combat ST131. PMID:24982321

  3. Recent Advances in Understanding Enteric Pathogenic Escherichia coli

    PubMed Central

    Croxen, Matthew A.; Law, Robyn J.; Scholz, Roland; Keeney, Kristie M.; Wlodarska, Marta

    2013-01-01

    SUMMARY Although Escherichia coli can be an innocuous resident of the gastrointestinal tract, it also has the pathogenic capacity to cause significant diarrheal and extraintestinal diseases. Pathogenic variants of E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide. Consequently, pathogenic E. coli is widely studied in humans, animals, food, and the environment. While there are many common features that these pathotypes employ to colonize the intestinal mucosa and cause disease, the course, onset, and complications vary significantly. Outbreaks are common in developed and developing countries, and they sometimes have fatal consequences. Many of these pathotypes are a major public health concern as they have low infectious doses and are transmitted through ubiquitous mediums, including food and water. The seriousness of pathogenic E. coli is exemplified by dedicated national and international surveillance programs that monitor and track outbreaks; unfortunately, this surveillance is often lacking in developing countries. While not all pathotypes carry the same public health profile, they all carry an enormous potential to cause disease and continue to present challenges to human health. This comprehensive review highlights recent advances in our understanding of the intestinal pathotypes of E. coli. PMID:24092857

  4. The Escherichia coli Proteome: Past, Present, and Future Prospects†

    PubMed Central

    Han, Mee-Jung; Lee, Sang Yup

    2006-01-01

    Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. PMID:16760308

  5. Fumarate-Mediated Persistence of Escherichia coli against Antibiotics

    PubMed Central

    Kim, Jun-Seob; Cho, Da-Hyeong; Heo, Paul; Jung, Suk-Chae; Park, Myungseo; Oh, Eun-Joong; Sung, Jaeyun; Kim, Pan-Jun; Lee, Suk-Chan; Lee, Dae-Hee; Lee, Sarah; Lee, Choong Hwan; Shin, Dongwoo

    2016-01-01

    Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) in Escherichia coli led to a higher frequency of persister formation. The persister frequency of E. coli was increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-related hipA7 mutation indicated that surplus fumarate markedly elevated the E. coli persister frequency. An E. coli strain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears that SDH and FRD represent a paired system that gives rise to and maintains E. coli persisters by producing and utilizing fumarate, respectively. PMID:26810657

  6. Escherichia coli β-Lactamases: What Really Matters

    PubMed Central

    Bajaj, Priyanka; Singh, Nambram S.; Virdi, Jugsharan S.

    2016-01-01

    Escherichia coli strains belonging to diverse pathotypes have increasingly been recognized as a major public health concern. The β-lactam antibiotics have been used successfully to treat infections caused by pathogenic E. coli. However, currently, the utility of β-lactams is being challenged severely by a large number of hydrolytic enzymes – the β-lactamases expressed by bacteria. The menace is further compounded by the highly flexible genome of E. coli, and propensity of resistance dissemination through horizontal gene transfer and clonal spread. Successful management of infections caused by such resistant strains requires an understanding of the diversity of β-lactamases, their unambiguous detection, and molecular mechanisms underlying their expression and spread with regard to the most relevant information about individual bacterial species. Thus, this review comprises first such effort in this direction for E. coli, a bacterial species known to be associated with production of diverse classes of β-lactamases. The review also highlights the role of commensal E. coli as a potential but under-estimated reservoir of β-lactamases-encoding genes. PMID:27065978

  7. Effects of Escherichia coli hemolysin on endothelial cell function.

    PubMed Central

    Suttorp, N; Flöer, B; Schnittler, H; Seeger, W; Bhakdi, S

    1990-01-01

    Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin. Images PMID:2121650

  8. The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

    PubMed Central

    Miskinyte, Migla; Sousa, Ana; Ramiro, Ricardo S.; de Sousa, Jorge A. Moura; Kotlinowski, Jerzy; Caramalho, Iris; Magalhães, Sara; Soares, Miguel P.; Gordo, Isabel

    2013-01-01

    Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity. PMID:24348252

  9. Unusual "flesh-eating" strains of Escherichia coli.

    PubMed

    Shaked, Hila; Samra, Zmira; Paul, Michal; Madar-Shapiro, Liora; Cohen, Jonathan; Pitlik, Silvio; Bishara, Jihad

    2012-12-01

    Monomicrobial necrotizing fasciitis (type II) is typically caused by group A streptococcus alone or in combination with Staphylococcus aureus. Escherichia coli has been isolated from polymicrobial or Fournier's gangrene but has rarely been reported in monomicrobial necrotizing fasciitis. We describe the clinical characteristics and outcomes of seven cases of monomicrobial E. coli necrotizing fasciitis and/or severe soft tissue infection diagnosed at a single institution during an 18-month period. Four isolates from three patients and two isolates from two patients with type I polymicrobial severe soft tissue infection (controls) were assayed by the randomly amplified polymorphic DNA (RAPD) analysis for fingerprinting and PCR amplification of primers in order to detect cytotoxic necrotizing factor 1 and 2 (cnf1 and cnf2) genes. All patients had some type of immune suppression. The limb was the most commonly involved organ. In all cases, E. coli was isolated as a monomicrobial pathogen from blood, fascia, or both. All patients died during hospitalization, three within the first 48 h. The RAPD amplification assay showed a high degree of genetic diversity among the "flesh-eating" strains and controls. The cnf1 toxin gene was identified in two out of three cases, but not in the controls. cnf2 was not detected in any of the patients. E. coli may be responsible for life-threatening necrotizing fasciitis. Further research is needed to reveal relevant risk factors, reservoirs, and modes of transmission of cnf1 E. coli.

  10. Escherichia coli exports cyclic AMP via TolC.

    PubMed

    Hantke, Klaus; Winkler, Karin; Schultz, Joachim E

    2011-03-01

    In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was sufficient to induce β-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce β-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

  11. Selective detection of Escherichia coli DNA using fluorescent carbon spindles.

    PubMed

    Roy, Anurag; Chatterjee, Sabyasachi; Pramanik, Srikrishna; Devi, Parukuttyamma Sujatha; Suresh Kumar, Gopinatha

    2016-04-28

    We investigate the interaction of hydrophilic blue emitting carbon spindles with various deoxyribonucleic acids (DNA) having different base pair compositions, such as Herring testes (HT), calf thymus (CT), Escherichia coli (EC) and Micrococcus lysodeikticus (ML) DNA, to understand the mode of interaction. Interestingly, the fluorescent carbon spindles selectively interacted with E. coli DNA resulting in enhanced fluorescence of the former. Interaction of the same carbon with other DNAs exhibited insignificant changes in fluorescence. In addition, in the presence of EC DNA, the D band in the Raman spectrum attributed to the defect state completely disappeared, resulting in enhanced crystallinity. Microscopy images confirmed the wrapping of DNA on the carbon spindles leading to the assembly of spindles in the form of flowers. Dissociation of double-stranded DNA occurred upon interaction with carbon spindles, resulting in selective E. coli DNA interaction. The carbon spindles also exhibited a similar fluorescence enhancement upon treating with E. coli bacteria. These results confirm the possibility of E. coli detection in water and other liquid foods using such fluorescent carbon.

  12. Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance

    PubMed Central

    Vega, Nicole M.; Allison, Kyle R.; Samuels, Amanda N.; Klempner, Mark S.; Collins, James J.

    2013-01-01

    Bacterial communication plays an important role in many population-based phenotypes and interspecies interactions, including those in host environments. These interspecies interactions may prove critical to some infectious diseases, and it follows that communication between pathogenic bacteria and commensal bacteria is a subject of growing interest. Recent studies have shown that Escherichia coli uses the signaling molecule indole to increase antibiotic tolerance throughout its population. Here, we show that the intestinal pathogen Salmonella typhimurium increases its antibiotic tolerance in response to indole, even though S. typhimurium does not natively produce indole. Increased antibiotic tolerance can be induced in S. typhimurium by both exogenous indole added to clonal S. typhimurium populations and indole produced by E. coli in mixed-microbial communities. Our data show that indole-induced tolerance in S. typhimurium is mediated primarily by the oxidative stress response and, to a lesser extent, by the phage shock response, which were previously shown to mediate indole-induced tolerance in E. coli. Further, we find that indole signaling by E. coli induces S. typhimurium antibiotic tolerance in a Caenorhabditis elegans model for gastrointestinal infection. These results suggest that the intestinal pathogen S. typhimurium can intercept indole signaling from the commensal bacterium E. coli to enhance its antibiotic tolerance in the host intestine. PMID:23946425

  13. Nonthermal atmospheric argon plasma jet effects on Escherichia coli biomacromolecules.

    PubMed

    Hosseinzadeh Colagar, Abasalt; Memariani, Hamed; Sohbatzadeh, Farshad; Valinataj Omran, Azadeh

    2013-12-01

    Nonthermal atmospheric plasma jet, a promising technology based on ionized gas at low temperatures, can be applied for disinfection of contaminated surfaces. In this study, Escherichia coli cells and their macromolecules were exposed to the nonthermal atmospheric argon plasma jet for different time durations. Total protein, genomic DNA, and malondialdehyde (MDA) levels of E. coli were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining; agarose gel electrophoresis; and measurement of absorbance at 534 nm, respectively. After exposure, the spectroscopic results of liquid samples indicated that the survival reduction of E. coli can reach to 100 % in an exposure time of 600 s. Moreover, inactivation zones of E. coli, DNA degradation, and MDA levels were significantly increased. Additionally, banding patterns of total protein were changed and amino acid concentrations increased following ninhydrin test. The experimental results suggest that the nonthermal plasma could serve as an effective instrument for both sterilizing E. coli and degrading macromolecules from the surface of the objects being sterilized.

  14. Paper-based ELISA to rapidly detect Escherichia coli.

    PubMed

    Shih, Cheng-Min; Chang, Chia-Ling; Hsu, Min-Yen; Lin, Jyun-Yu; Kuan, Chen-Meng; Wang, Hsi-Kai; Huang, Chun-Te; Chung, Mu-Chi; Huang, Kui-Chou; Hsu, Cheng-En; Wang, Chun-Yuan; Shen, Ying-Cheng; Cheng, Chao-Min

    2015-12-01

    Escherichia coli is a generic indicator of fecal contamination, and certain serotypes cause food- and water-borne illness such as O157:H7. In the clinic, detection of bacteriuria, which is often due to E. coli, is critical before certain surgical procedures or in cases of nosocomial infection to prevent further adverse events such as postoperative infection or sepsis. In low- and middle-income countries, where insufficient equipment and facilities preclude modern methods of detection, a simple, low-cost diagnostic device to detect E. coli in water and in the clinic will have significant impact. We have developed a simple paper-based colorimetric platform to detect E. coli contamination in 5h. On this platform, the mean color intensity for samples with 10(5)cells/mL is 0.118±0.002 (n=4), and 0.0145±0.003 (P<0.01⁎⁎) for uncontaminated samples. This technique is less time-consuming, easier to perform, and less expensive than conventional methods. Thus, paper-based ELISA is an innovative point-of-care diagnostic tool to rapidly detect E. coli, and possibly other pathogens when customized as appropriate, especially in areas that lack advanced clinical equipment.

  15. Pulsed-Plasma Disinfection of Water Containing Escherichia coli

    NASA Astrophysics Data System (ADS)

    Satoh, Kohki; MacGregor, Scott J.; Anderson, John G.; Woolsey, Gerry A.; Fouracre, R. Anthony

    2007-03-01

    The disinfection of water containing the microorganism, Escherichia coli (E. coli) by exposure to a pulsed-discharge plasma generated above the water using a multineedle electrode (plasma-exposure treatment), and by sparging the off-gas of the pulsed plasma into the water (off-gas-sparging treatment), is performed in the ambient gases of air, oxygen, and nitrogen. For the off-gas-sparging treatment, bactericidal action is observed only when oxygen is used as the ambient gas, and ozone is found to generate the bactericidal action. For the plasma-exposure treatment, the density of E. coli bacteria decreases exponentially with plasma-exposure time for all the ambient gases. It may be concluded that the main contributors to E. coli inactivation are particle species produced by the pulsed plasma. For the ambient gases of air and nitrogen, the influence of acidification of the water in the system, as a result of pulsed-plasma exposure, may also contribute to the decay of E. coli density.

  16. Susceptibility of Gnotobiotic Swine to Escherichia coli Isolated from Nonenteric Human Infections

    PubMed Central

    Meyer, R. C.; Rhoades, H. E.; Simon, J.

    1972-01-01

    Newborn, germfree piglets were susceptible to Escherichia coli associated with human, nonenteric infections and should provide a useful model in the study of generalized E. coli infections. PMID:4557565

  17. Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes.

    PubMed

    Kessler, Robert; Nisa, Shahista; Hazen, Tracy H; Horneman, Amy; Amoroso, Anthony; Rasko, David A; Donnenberg, Michael S

    2015-11-01

    A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli.

  18. Discovery of Fur binding site clusters in Escherichia coli by information theory models

    PubMed Central

    Chen, Zehua; Lewis, Karen A.; Shultzaberger, Ryan K.; Lyakhov, Ilya G.; Zheng, Ming; Doan, Bernard; Storz, Gisela; Schneider, Thomas D.

    2007-01-01

    Fur is a DNA binding protein that represses bacterial iron uptake systems. Eleven footprinted Escherichia coli Fur binding sites were used to create an initial information theory model of Fur binding, which was then refined by adding 13 experimentally confirmed sites. When the refined model was scanned across all available footprinted sequences, sequence walkers, which are visual depictions of predicted binding sites, frequently appeared in clusters that fit the footprints (∼83% coverage). This indicated that the model can accurately predict Fur binding. Within the clusters, individual walkers were separated from their neighbors by exactly 3 or 6 bases, consistent with models in which Fur dimers bind on different faces of the DNA helix. When the E. coli genome was scanned, we found 363 unique clusters, which includes all known Fur-repressed genes that are involved in iron metabolism. In contrast, only a few of the known Fur-activated genes have predicted Fur binding sites at their promoters. These observations suggest that Fur is either a direct repressor or an indirect activator. The Pseudomonas aeruginosa and Bacillus subtilis Fur models are highly similar to the E. coli Fur model, suggesting that the Fur–DNA recognition mechanism may be conserved for even distantly related bacteria. PMID:17921503

  19. Triphenyltin degradation and proteomic response by an engineered Escherichia coli expressing cytochrome P450 enzyme.

    PubMed

    Yi, Wenying; Yang, Kunliang; Ye, Jinshao; Long, Yan; Ke, Jing; Ou, Huase

    2017-03-01

    Although triphenyltin (TPT) degradation pathway has been determined, information about the enzyme and protein networks involved was severely limited. To this end, a cytochrome P450 hydroxylase (CYP450) gene from Bacillus thuringiensis was cloned and expressed in Escherichia coli BL21 (DE3), namely E. coli pET32a-CYP450, whose dosage at 1gL(-1) could degrade 54.6% TPT at 1mgL(-1) within 6 d through attacking the carbon-tin bonds of TPT by CYP450. Sequence analysis verified that the CYP450 gene had a 1214bp open reading frame, encoding a protein with 404 amino acids. Proteomic analysis determined that 60 proteins were significantly differentially regulated expression in E. coli pET32a-CYP450 after TPT degradation. The up-regulated proteins enriched in a network related to transport, cell division, biosynthesis of amino acids and secondary metabolites, and microbial metabolism in diverse environments. The current findings demonstrated for the first time that P450 received electrons transferring from NADH could effectively cleave carbon-metal bonds.

  20. Multidrug-resistant Escherichia coli soft tissue infection investigated with bacterial whole genome sequencing

    PubMed Central

    Buchanan, Ruaridh; Stoesser, Nicole; Crook, Derrick; Bowler, Ian C J W

    2014-01-01

    A 45-year-old man with dilated cardiomyopathy presented with acute leg pain and erythema suggestive of necrotising fasciitis. Initial surgical exploration revealed no necrosis and treatment for a soft tissue infection was started. Blood and tissue cultures unexpectedly grew a Gram-negative bacillus, subsequently identified by an automated broth microdilution phenotyping system as an extended-spectrum β-lactamase producing Escherichia coli. The patient was treated with a 3-week course of antibiotics (ertapenem followed by ciprofloxacin) and debridement for small areas of necrosis, followed by skin grafting. The presence of E. coli triggered investigation of both host and pathogen. The patient was found to have previously undiagnosed liver disease, a risk factor for E. coli soft tissue infection. Whole genome sequencing of isolates from all specimens confirmed they were clonal, of sequence type ST131 and associated with a likely plasmid-associated AmpC (CMY-2), several other resistance genes and a number of virulence factors. PMID:25331151

  1. A moonlighting enzyme links Escherichia coli cell size with central metabolism.

    PubMed

    Hill, Norbert S; Buske, Paul J; Shi, Yue; Levin, Petra Anne

    2013-01-01

    Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.

  2. A modified pathway for the production of acetone in Escherichia coli.

    PubMed

    May, Antje; Fischer, Ralf-Jörg; Maria Thum, Simone; Schaffer, Steffen; Verseck, Stefan; Dürre, Peter; Bahl, Hubert

    2013-01-01

    A modified synthetic acetone operon was constructed. It consists of two genes from Clostridium acetobutylicum (thlA coding for thiolase and adc coding for acetoacetate decarboxylase) and one from Bacillus subtilis or Haemophilus influenzae (teII(srf) or ybgC, respectively, for thioesterase). Expression of this operon in Escherichia coli resulted in the production of acetone starting from the common metabolite acetyl-CoA via acetoacetyl-CoA and acetoacetate. The thioesterases do not need a CoA acceptor for acetoacetyl-CoA hydrolysis. Thus, in contrast to the classic acetone pathway of Clostridium acetobutylicum and related microorganisms which employ a CoA transferase, the new pathway is acetate independent. The genetic background of the host strains was crucial. Only E. coli strains HB101 and WL3 were able to produce acetone via the modified plasmid based pathway, up to 64mM and 42mM in 5-ml cultures, respectively. Using glucose fed-batch cultures the concentration could be increased up to 122mM acetone with HB101 carrying the recombinant plasmid pUC19ayt (thioesterase from H. influenzae). The formation of acetone led to a decreased acetate production by E. coli.

  3. Role of enteroaggregative Escherichia coli virulence factors in uropathogenesis.

    PubMed

    Boll, Erik J; Struve, Carsten; Boisen, Nadia; Olesen, Bente; Stahlhut, Steen G; Krogfelt, Karen A

    2013-04-01

    A multiresistant clonal Escherichia coli O78:H10 strain qualifying molecularly as enteroaggregative Escherichia coli (EAEC) was recently shown to be the cause of a community-acquired outbreak of urinary tract infection (UTI) in greater Copenhagen, Denmark, in 1991. This marks the first time EAEC has been associated with an extraintestinal disease outbreak. Importantly, the outbreak isolates were recovered from the urine of patients with symptomatic UTI, strongly implying urovirulence. Here, we sought to determine the uropathogenic properties of the Copenhagen outbreak strain and whether these properties are conferred by the EAEC-specific virulence factors. We demonstrated that through expression of aggregative adherence fimbriae, the principal adhesins of EAEC, the outbreak strain exhibited pronouncedly increased adherence to human bladder epithelial cells compared to prototype uropathogenic strains. Moreover, the strain was able to produce distinct biofilms on abiotic surfaces, including urethral catheters. These findings suggest that EAEC-specific virulence factors increase uropathogenicity and may have played a significant role in the ability of the strain to cause a community-acquired outbreak of UTI. Thus, inclusion of EAEC-specific virulence factors is warranted in future detection and characterization of uropathogenic E. coli.

  4. Genotyping and virulence factors assessment of bovine mastitis Escherichia coli.

    PubMed

    Blum, Shlomo E; Leitner, Gabriel

    2013-05-03

    Escherichia coli is a major agent of bovine mastitis worldwide. However, specific E. coli virulence factors associated to pathogenicity during intra-mammary infections are yet unknown and this pathotype remains uncharacterized. The objectives of the present work were to assess the presence of a wide range of known virulence factors in a large set of E. coli strains isolated from bovine mastitis (mastitis set) and to study the genotypic distribution of strains in the mastitis set in comparison to a set of strains isolated from cows' environment in dairy farms (environmental set). Virulence factors were assessed by DNA hybridization microarray. The three most prevalent virulence factors found in the mastitis set were lpfA (long polar fimbriae), iss (increased serum resistance) and astA (enteroaggregative E. coli heat-stable enterotoxin 1). None, however, characterized the majority of these strains. Genotyping was assessed by ECOR phylogenetic grouping, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Strains in the mastitis and environmental sets were differentially distributed into ECOR phylogenetic groups; groups A and B1 being the most prevalent ones. Multiple MLST strain types were found in the two sets of strains, but only a few were common to both, and diversity was higher in the environmental set. A variety of PFGE patterns were found in the mastitis and environmental sets. Two clusters comprising mostly highly similar mastitis strains were identified. The results confirm that mastitis E. coli strains mostly lack known E. coli virulence factors. In addition, it is shown that the genotypic diversity of mastitis strains does not reflect the diversity found in the environmental E. coli population.

  5. Distribution of core oligosaccharide types in lipopolysaccharides from Escherichia coli.

    PubMed

    Amor, K; Heinrichs, D E; Frirdich, E; Ziebell, K; Johnson, R P; Whitfield, C

    2000-03-01

    In the lipopolysaccharides of Escherichia coli there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the waa (core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of E. coli. This system was applied to the 72 isolates in the E. coli ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69. 4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic E. coli isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal E. coli isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic E. coli (VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic E. coli serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.

  6. Reduction of verotoxigenic Escherichia coli in production of fermented sausages.

    PubMed

    Holck, Askild L; Axelsson, Lars; Rode, Tone Mari; Høy, Martin; Måge, Ingrid; Alvseike, Ole; L'abée-Lund, Trine M; Omer, Mohamed K; Granum, Per Einar; Heir, Even

    2011-11-01

    After a number of foodborne outbreaks of verotoxigenic Escherichia coli involving fermented sausages, some countries have imposed regulations on sausage production. For example, the US Food Safety and Inspection Service requires a 5 log(10) reduction of E. coli in fermented products. Such regulations have led to a number of studies on the inactivation of E. coli in fermented sausages by changing processing and post-processing conditions. Several factors influence the survival of E. coli such as pre-treatment of the meat, amount of NaCl, nitrite and lactic acid, water activity, pH, choice of starter cultures and addition of antimicrobial compounds. Also process variables like fermentation temperature and storage time play important roles. Though a large variety of different production processes of sausages exist, generally the reduction of E. coli caused by production is in the range 1-2 log(10). In many cases this may not be enough to ensure microbial food safety. By optimising ingredients and process parameters it is possible to increase E. coli reduction to some extent, but in some cases still other post process treatments may be required. Such treatments may be storage at ambient temperatures, specific heat treatments, high pressure processing or irradiation. HACCP analyses have identified the quality of the raw materials, low temperature in the batter when preparing the sausages and a rapid pH drop during fermentation as critical control points in sausage production. This review summarises the literature on the reduction verotoxigenic E. coli in production of fermented sausages.

  7. Persistence of Escherichia coli in batch and continuous vermicomposting systems.

    PubMed

    Hénault-Ethier, Louise; Martin, Vincent J J; Gélinas, Yves

    2016-10-01

    Vermicomposting is a biooxidation process in which epigeicearthworms act in synergy with microbial populations to degrade organic matter. Vermicomposting does not go through a thermophilic stage as required by North American legislations for pathogen eradication. We examined the survival of a Green Fluorescent Protein (GFP) labeled Escherichia coli MG1655 as a model for the survival of pathogenic bacteria in both small-scale batch and medium-scale continuously-operated systems to discern the influence of the earthworm Eisenia fetida, nutrient content and the indigenous vermicompost microbial community on pathogen abundance. In batch systems, the microbial community had the greatest influence on the rapid decline of E. coli populations, and the effect of earthworms was only visible in microbially-impoverishedvermicomposts. No significant earthworm density-dependent relationship was observed on E. coli survival under continuous operation. E. coli numbers decreased below the US EPA compost sanitation guidelines of 10(3)Colony Forming Units (CFU)/g (dry weight) within 18-21days for both the small-scale batch and medium-scale continuous systems, but it took up to 51days without earthworms and with an impoverished microbial community to reach the legal limit. Nutrient replenishment (i.e. organic carbon) provided by continuous feed input did not appear to extend E. coli survival. In fact, longer survival of E. coli was noticed in treatments where less total and labile sugars were available, suggesting that sugars may support potentially antagonist bacteria in the vermicompost. Total N, pH and humidity did not appear to affect E. coli survival. Several opportunistic human pathogens may be found in vermicompost, and their populations are likely kept in check by antagonists.

  8. Diarrhea, Urosepsis and Hemolytic Uremic Syndrome Caused by the Same Heteropathogenic Escherichia coli Strain.

    PubMed

    Ang, C Wim; Bouts, Antonia H M; Rossen, John W A; Van der Kuip, Martijn; Van Heerde, Marc; Bökenkamp, Arend

    2016-09-01

    We describe an 8-month-old girl with diarrhea, urosepsis and hemolytic uremic syndrome caused by Escherichia coli. Typing of cultured E. coli strains from urine and blood revealed the presence of virulence factors from multiple pathotypes of E. coli. This case exemplifies the genome plasticity of E. coli and the resulting heteropathogenic strains.

  9. Genome Sequence of the Enterohemorrhagic Escherichia coli Bacteriophage UFV-AREG1.

    PubMed

    Lopez, Maryoris E Soto; Batalha, Laís Silva; Vidigal, Pedro Marcus Pereira; Albino, Luiz Augusto A; Boggione, Delaine Meireles Gouveia; Gontijo, Marco Tulio Pardini; Bazzolli, Denise M Soares; Mendonca, Regina C Santos

    2016-10-13

    Here, we present the genome sequence of the Escherichia coli bacteriophage UFV-AREG1. This phage was isolated from cowshed wastewater and showed specificity for enterohemorrhagic E. coli O157:H7 (ATCC 43895), E. coli 0111 (CDC O11ab) and E. coli (ATCC 23229).

  10. Genome Sequence of the Enterohemorrhagic Escherichia coli Bacteriophage UFV-AREG1

    PubMed Central

    Batalha, Laís Silva; Albino, Luiz Augusto A.; Boggione, Delaine Meireles Gouveia; Gontijo, Marco Tulio Pardini; Bazzolli, Denise M. Soares; Mendonca, Regina C. Santos

    2016-01-01

    Here, we present the genome sequence of the Escherichia coli bacteriophage UFV-AREG1. This phage was isolated from cowshed wastewater and showed specificity for enterohemorrhagic E. coli O157:H7 (ATCC 43895), E. coli 0111 (CDC O11ab) and E. coli (ATCC 23229). PMID:27738021

  11. Multiplex PCR for Diagnosis of Enteric Infections Associated with Diarrheagenic Escherichia coli

    PubMed Central

    Vidal, Roberto; Vidal, Maricel; Lagos, Rossana; Levine, Myron; Prado, Valeria

    2004-01-01

    A multiplex PCR for detection of three categories of diarrheagenic Escherichia coli was developed. With this method, enterohemorrhagic E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were identified in fecal samples from patients with hemorrhagic colitis, watery diarrhea, or hemolytic-uremic syndrome and from food-borne outbreaks. PMID:15071051

  12. Persistent colonization of sheep by Escherichia coli O157:H7 and other E. coli pathotypes.

    PubMed

    Cornick, N A; Booher, S L; Casey, T A; Moon, H W

    2000-11-01

    Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10(7) or 10(10) CFU/strain/animal. The other strains were given only at 10(10) CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10(7) or 10(10) CFU. One of the ETEC strains also persisted when inoculated at 10(10) CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10(7) CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.

  13. Effect of bile on growth, peritoneal absorption, and blood clearance of Escherichia coli in E coli peritonitis

    SciTech Connect

    Andersson, R.; Schalen, C.; Tranberg, K.G. )

    1991-06-01

    The effect of intraperitoneal bile on growth, peritoneal absorption, and clearance of Escherichia coli was determined in E coli peritonitis in the rat. In E coli peritonitis, intraperitoneal bacterial counts gradually decreased, whereas they increased (after 2 hours) with subsequent development of bacteremia in E coli plus bile peritonitis. After an intraperitoneal injection of labeled bacteria, blood radioactivity was only initially lower in E coli plus bile peritonitis compared with E coli peritonitis. Clearance from blood was lower in E coli plus bile peritonitis than in E coli peritonitis. Organ localization was similar in E coli peritonitis and E coli plus bile peritonitis with decreased splenic, increased pulmonary, and unchanged hepatic uptakes compared with controls. Impaired peritoneal absorption of bacteria, together with impaired local host defense, is likely to enhance the noxious effect of bile in E coli peritonitis.

  14. Structure of the Escherichia coli S10 ribosomal protein operon.

    PubMed Central

    Zurawski, G; Zurawski, S M

    1985-01-01

    The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data. PMID:3892488

  15. Genome-scale genetic engineering in Escherichia coli.

    PubMed

    Jeong, Jaehwan; Cho, Namjin; Jung, Daehee; Bang, Duhee

    2013-11-01

    Genome engineering has been developed to create useful strains for biological studies and industrial uses. However, a continuous challenge remained in the field: technical limitations in high-throughput screening and precise manipulation of strains. Today, technical improvements have made genome engineering more rapid and efficient. This review introduces recent advances in genome engineering technologies applied to Escherichia coli as well as multiplex automated genome engineering (MAGE), a recent technique proposed as a powerful toolkit due to its straightforward process, rapid experimental procedures, and highly efficient properties.

  16. Antitermination of transcription from an Escherichia coli ribosomal RNA promoter.

    PubMed

    Holben, W E; Morgan, E A

    1984-11-01

    The Escherichia coli lac and ara promoters and rrnC ribosomal RNA promoter-leader region were fused to lacZYA. Transcription termination signals were introduced into the lac genes of these fusions by Tn9 and IS1 insertions. Measurement of lac enzymes from upstream and downstream of the insertions showed that termination signals resulting from these insertions are very efficient when transcription begins at lac or ara promoters but are very inefficient when transcription begins at the rrnC promoter-leader region. The rrnC promoter-leader region must, therefore, modify RNA polymerase to enable it to read through transcription termination signals.

  17. Regulation of the L-arabinose operon of Escherichia coli.

    PubMed

    Schleif, R

    2000-12-01

    Over forty years of research on the L-arabinose operon of Escherichia coli have provided insights into the mechanism of positive regulation of gene activity. This research also discovered DNA looping and the mechanism by which the regulatory protein changes its DNA-binding properties in response to the presence of arabinose. As is frequently seen in focused research on biological subjects, the initial studies were primarily genetic. Subsequently, the genetic approaches were augmented by physiological and then biochemical studies. Now biophysical studies are being conducted at the atomic level, but genetics still has a crucial role in the study of this system.

  18. Studies on the Chick-lethal Toxin of Escherichia coli

    PubMed Central

    Truscott, R. B.

    1973-01-01

    A toxin which is lethal for two week old chicks has been recovered from strains of Escherichia coli O78:K80 of bovine and avian origin and from avian isolates of serogroups O2, O45 and O109. The toxin is heat-labile, antigenic, high in protein, inactivated by pronase, trypsin, amylase, and pancreatic lipase. The toxin may be precipitated by ammonium sulfate or TCA treatment from the supernatant obtained by repeated centrifugation of sonicated cells. Considerable purification has been obtained by column chromatography using Sepharose 6B. PMID:4270809

  19. Compilation and analysis of Escherichia coli promoter DNA sequences.

    PubMed Central

    Hawley, D K; McClure, W R

    1983-01-01

    The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence. PMID:6344016

  20. [Hemolytic uremic syndrome caused by enterohaemorrhagic Escherichia coli].

    PubMed

    Ibarra, Cristina; Goldstein, Jorge; Silberstein, Claudia; Zotta, Elsa; Belardo, Marcela; Repetto, Horacio A

    2008-10-01

    Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, plaquetopenia and kidney damage. It is the leading cause of acute renal failure in pediatric age and the second for chronic renal failure. Shiga toxin-producing Escherichia coli (STEC) is the first etiologic agent of HUS being its main reservoir cattle and transmitted via contaminated food. At present, there is no specific treatment to reduce the progression of HUS. The study of the mechanisms by which STEC infects and Shiga toxin induces HUS can help to find new strategies to prevent this disease.

  1. Microcin 25, a novel antimicrobial peptide produced by Escherichia coli.

    PubMed Central

    Salomón, R A; Farías, R N

    1992-01-01

    Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system. Images PMID:1429464

  2. Interaction of the exr and lon Genes in Escherichia coli

    PubMed Central

    Donch, John; Green, Michael H. L.; Greenberg, Joseph

    1968-01-01

    Strains of Escherichia coli carrying the gene lon typically produced excess capsular polysaccharide, and were sensitive to ultraviolet light (UV) irradiation, thymine starvation, and nalidixic acid, forming long filaments after these treatments. Sensitivity was reduced by a number of posttreatments. In the presence of a second UV sensitivity gene, exr, some of these properties were suppressed: long filaments were not formed, the effect of lon on UV and nalidixic acid sensitivity was greatly reduced, and irradiation posttreatments gave an enhancement of survival characteristic of exr rather than lon strains. Production of capsular polysaccharide was not affected by the exr gene. PMID:4882020

  3. CRISPR adaptation in Escherichia coli subtypeI-E system.

    PubMed

    Kiro, Ruth; Goren, Moran G; Yosef, Ido; Qimron, Udi

    2013-12-01

    The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called 'spacers', which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed 'adaptation'. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR-Cas system of Escherichia coli.

  4. Synthesis of calf prochymosin (prorennin) in Escherichia coli.

    PubMed Central

    Emtage, J S; Angal, S; Doel, M T; Harris, T J; Jenkins, B; Lilley, G; Lowe, P A

    1983-01-01

    A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach. Images PMID:6304731

  5. DNA probes for identification of enteroinvasive Escherichia coli.

    PubMed Central

    Gomes, T A; Toledo, M R; Trabulsi, L R; Wood, P K; Morris, J G

    1987-01-01

    Eighty-one Escherichia coli strains belonging to all known invasive O serogroups were tested with two distinct invasiveness probes (pMR17 and pSF55). All 54 Sereny test-positive strains and 5 strains that lost Sereny positivity during storage hybridized with both probes. Probe-positive strains carried a 120- to 140-megadalton plasmid, did not produce lysine decarboxylase, and, with the exception of certain serotypes, were nonmotile. Motile strains of serotype O144:H25 were for the first time characterized as invasive by hybridization with the probes. PMID:3312292

  6. Electric dipole moments of Escherichia coli HB 101.

    PubMed

    Stoylov, Stoyl P; Gyurova, Anna Y; Bunin, Viktor; Angersbach, Alexander; Georgieva, Ralitsa N; Danova, Svetla T

    2009-04-01

    The theoretical and experimental studies of the particles' electric dipole moments in the microscopic and submicroscopic size range show that in the case of polar and conductive media the interfacial components of the dipole moments are of greatest importance. While in the range of manometer's sizes there seems to be no important problems in the identification and in the estimation of the values of the dipole moments at present, in the micrometer range there are serious problems. In this communication these problems are considered and illustrated by electro-optic investigations of Escherichia coli HB 101.

  7. Infected abdominal aortic aneurysm due to Escherichia coli.

    PubMed

    Bouzas, Miguel; Tchana-Sato, Vincent; Lavigne, Jean Paul

    2016-10-19

    Early diagnosis of infected abdominal aortic aneurysm (IAAA) is still a medical challenge due to its diverse and non-specific symptoms and signs. The most common responsible pathogens are Salmonella, Staphylococcus, Campylobacter and Streptococcus species. The authors report the case of a 67-year-old man, admitted for high fever and finally diagnosed with Escherichia coli (E.coli)-related IAAA. The IAAA ruptured during the general anaesthesia induction, leading to an emergency surgery. The authors successfully proceeded to an open aneurysmectomy with extensive debridement and in situ graft replacement. This case emphasizes the potential for rapid IAAA expansion, its high-rupture risk and the importance of computed tomography as a diagnostic tool.

  8. Impact of cranberry on Escherichia coli cellular surface characteristics

    SciTech Connect

    Johnson, Brandy J.; Malanoski, Anthony P.; Ligler, Frances S.

    2008-12-19

    The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

  9. Avian pathogenic Escherichia coli bind fibronectin and laminin.

    PubMed

    Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

    2009-04-01

    Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

  10. Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase.

    PubMed

    Kantrowitz, Evan R

    2012-03-15

    The allosteric enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli has been the subject of investigations for approximately 50 years. This enzyme controls the rate of pyrimidine nucleotide biosynthesis by feedback inhibition, and helps to balance the pyrimidine and purine pools by competitive allosteric activation by ATP. The catalytic and regulatory components of the dodecameric enzyme can be separated and studied independently. Many of the properties of the enzyme follow the Monod, Wyman Changeux model of allosteric control thus E. coli ATCase has become the textbook example. This review will highlight kinetic, biophysical, and structural studies which have provided a molecular level understanding of how the allosteric nature of this enzyme regulates pyrimidine nucleotide biosynthesis.

  11. Purification of recombinant ovalbumin from inclusion bodies of Escherichia coli.

    PubMed

    Upadhyay, Vaibhav; Singh, Anupam; Panda, Amulya K

    2016-01-01

    Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications.

  12. Inversions between ribosomal RNA genes of Escherichia coli.

    PubMed Central

    Hill, C W; Harnish, B W

    1981-01-01

    It might be anticipated that the presence of redundant but oppositely oriented sequences in a chromosome could allow inversion of the intervening material through homologous recombination. For example, the ribosomal RNA gene rrnD of Escherichia coli has the opposite orientation fro rrnB and rrnE and is separated from these genes by roughly 20% of the chromosome. Starting with a derivative of Cavalli Hfr, we have constructed mutants that have an inversion of the segment between rrnD and either rrnB or rrnE. These mutants are generally quite viable but do exhibit a slight reduction in growth rate relative to the parental strain. A major line of laboratory E. coli, W3110 and its derivatives, also has an inversion between rrnD and rrnE, probably created directly by a recombinational event between these highly homologous genes. Images PMID:6273909

  13. SILVER NANOPARTICLES-DISK DIFFUSION TEST AGAINST Escherichia coli ISOLATES.

    PubMed

    Cunha, Francisco Afrânio; Maia, Kamila Rocha; Mallman, Eduardo José Jucá; Cunha, Maria da Conceição Dos Santos Oliveira; Maciel, Antonio Auberson Martins; Souza, Ieda Pereira de; Menezes, Everardo Albuquerque; Fechine, Pierre Basílio Almeida

    2016-09-22

    Nanotechnology can be a valuable ally in the treatment of infections. Silver nanoparticles (AgNPs) are structures that have antimicrobial activity. The aim of this study was to produce AgNPs by green methods, characterize these structures, and assess their antimicrobial activity against Escherichia coli associated with the antibiotic ciprofloxacin. AgNPs were characterized by spectroscopic and microscopic techniques. Antimicrobial activity was evaluated by the disk diffusion method against 10 strains of E. coli. The synthesized AgNPs showed a spherical shape and a size of 85.07 ± 12.86 nm (mean ± SD). AgNPs increased the activity of ciprofloxacin by 40% and may represent a new therapeutic option for the treatment of bacterial infections.

  14. Continuous-sterilization system that uses photosemiconductor powders. [Escherichia coli

    SciTech Connect

    Matsunaga, T.; Tomoda, R.; Nakajima, T.; Nakamura, N.; Komine, T.

    1988-06-01

    We report a novel photochemical sterilization system in which Escherichia coli cells were sterilized with photosemiconductor powders (titanium oxide). For sterilization that could be used in practice, it was necessary to separate the TiO/sub 2/ powders from the cell suspension. Therefore, semiconductor powders were immobilized on acetylcellulose membranes. We constructed a continuous-sterilization system consisting of TiO/sub 2/-immobilized acetylcellulose membrane reactor, a mercury lamp, and a masterflex pump. As a result, under the various sterilization conditions examined, E.coli (10/sup 2/ cells per ml) was sterilized to < 1% survival when the cell suspension flowed in this system at a mean residence time of 16.0 min under irradiation (1800 microeinsteins/m/sup 2/ per s). We found that this system was reusable.

  15. Detecting the Significant Flux Backbone of Escherichia coli metabolism.

    PubMed

    Güell, Oriol; Sagués, Francesc; Serrano, M Ángeles

    2017-04-09

    The heterogeneity of computationally predicted reaction fluxes in metabolic networks within a single flux state can be exploited to detect their significant flux backbone. Here, we disclose the backbone of Escherichia coli, and compare it with the backbones of other bacteria. We find that, in general, the core of the backbones is mainly composed of reactions in energy metabolism corresponding to ancient pathways. In E. coli, the synthesis of nucleotides and the metabolism of lipids form smaller cores which rely critically on energy metabolism. Moreover, the consideration of different media leads to the identification of pathways sensitive to environmental changes. The metabolic backbone of an organism is thus useful for tracing, simultaneously, both its evolution and adaptation fingerprints. This article is protected by copyright. All rights reserved.

  16. The SIGNAL experiment in BIORACK: Escherichia coli in microgravity.

    PubMed

    Thévenet, D; D'Ari, R; Bouloc, P

    1996-06-27

    Microgravity affects certain physical properties of fluids, such as convection movement and surface tension. As a consequence, cells and living organisms may exhibit different behaviour in space, which may result from differences in the immediate environment of the cell or changes in the structure of the membrane in microgravity. Two experiments to examine the effects of microgravity on cell microenvironment and signal transduction through membranes were performed using a well-characterized system with different strains of the non-pathogenic Gram-negative bacterium Escherichia coli. Our results indicate that (i) microgravity appears to reduce the lag period of a non-motile culture of E. coli, and (ii) the ompC gene, regulated by the two-component system EnvZ-OmpR, is induced as well or better in microgravity than in ground controls.

  17. Mounting of Escherichia coli spheroplasts for AFM imaging.

    SciTech Connect

    Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P; Doktycz, Mitchel John

    2005-11-01

    The cytoplasmic membrane of Escherichia coli (E. coli) is the location of numerous, chemically specific transporters and recognition elements. Investigation of this membrane in vivo by atomic force microscopy (AFM) requires removal of the cell wall and stable immobilization of the spheroplast. AFM images demonstrate that spheroplasts can be secured with warm gelatin applied to the mica substrate just before the addition of a spheroplast suspension. The resulting preparation can be repeatedly imaged by AFM over the course of several hours. Confocal fluorescence imaging confirms the association of the spheroplasts with the gelatin layer. Gelatin molecules are known to reorder into a network after heating. Entrapment within this gelatin network is believed to be responsible for the immobilization of spheroplasts on mica.

  18. The action of beta-galactosidase (Escherichia coli) on allolactose.

    PubMed

    Huber, R E; Wallenfels, K; Kurz, G

    1975-09-01

    The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.

  19. In Vivo study of naturally deformed Escherichia coli bacteria.

    PubMed

    Tavaddod, Sharareh; Naderi-Manesh, Hossein

    2016-06-01

    A combination of light-microscopy and image processing has been applied to study naturally deformed Escherichia coli under in vivo condition and at the order of sub-pixel high-resolution accuracy. To classify deflagellated non-dividing E. coli cells to the rod-shape and bent-shape, a geometrical approach has been applied. From the analysis of the geometrical data which were obtained of image processing, we estimated the required effective energy for shaping a rod-shape to a bent-shape with the same size. We evaluated the energy of deformation in the naturally deformed bacteria with minimum cell manipulation, under in vivo condition, and with minimum influence of any external force, torque and pressure. Finally, we have also elaborated on the possible scenario to explain how naturally deformed bacteria are formed from initial to final-stage.

  20. SILVER NANOPARTICLES-DISK DIFFUSION TEST AGAINST Escherichia coli ISOLATES

    PubMed Central

    CUNHA, Francisco Afrânio; MAIA, Kamila Rocha; MALLMAN, Eduardo José Jucá; CUNHA, Maria da Conceição dos Santos Oliveira; MACIEL, Antonio Auberson Martins; de SOUZA, Ieda Pereira; MENEZES, Everardo Albuquerque; FECHINE, Pierre Basílio Almeida

    2016-01-01

    SUMMARY Nanotechnology can be a valuable ally in the treatment of infections. Silver nanoparticles (AgNPs) are structures that have antimicrobial activity. The aim of this study was to produce AgNPs by green methods, characterize these structures, and assess their antimicrobial activity against Escherichia coli associated with the antibiotic ciprofloxacin. AgNPs were characterized by spectroscopic and microscopic techniques. Antimicrobial activity was evaluated by the disk diffusion method against 10 strains of E. coli. The synthesized AgNPs showed a spherical shape and a size of 85.07 ± 12.86 nm (mean ± SD). AgNPs increased the activity of ciprofloxacin by 40% and may represent a new therapeutic option for the treatment of bacterial infections. PMID:27680178

  1. Mutational analysis of UMP kinase from Escherichia coli.

    PubMed

    Bucurenci, N; Serina, L; Zaharia, C; Landais, S; Danchin, A; Bârzu, O

    1998-02-01

    UMP kinase from Escherichia coli is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of E. coli UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).

  2. Identification, expression, and characterization of Escherichia coli guanine deaminase.

    PubMed

    Maynes, J T; Yuan, R G; Snyder, F F

    2000-08-01

    Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease.

  3. Identification, Expression, and Characterization of Escherichia coli Guanine Deaminase

    PubMed Central

    Maynes, Jason T.; Yuan, Richard G.; Snyder, Floyd F.

    2000-01-01

    Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a Km of 15 μM with guanine and a kcat of 3.2 s−1. The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3′ from an open reading frame which shows homology to a bacterial purine base permease. PMID:10913105

  4. Effect of various nonionic surfactants on growth of Escherichia coli.

    PubMed

    Rose, M J; Aron, S A; Janicki, B W

    1966-05-01

    Rose, Michael J., Jr. (Veterans Administration Hospital, Washington, D.C.), Stephen A. Aron, and Bernard W. Janicki. Effect of various nonionic surfactants on growth of Escherichia coli. J. Bacteriol. 91:1863-1868. 1966.-Escherichia coli cultivated in media containing 0.5, 1.0, 2.0, or 4.0% concentrations of surface-active polyoxyethylene derivatives of formaldehyde polymers of octyl phenol (Triton WR-1339; Macrocyclon) or of sorbitan mono-fatty acid esters (Tween 20, 40, 60, and 80) exhibited significantly retarded growth only at the highest concentration. To determine the mechanism of bacteriostasis, certain derivatives and compounds related to the surfactants were investigated. Experiments with compounds related to the Triton-type agents demonstrated that incorporation of monomeric substances (Triton X-205, X-305, Igepal CA-730, or Dowfax 9N20) into the medium at a concentration of 4.0% did not inhibit the growth of E. coli. It was concluded that the formaldehyde polymer was essential for growth inhibition by the polyoxyethylene derivatives of octyl phenol. The inhibitory activity of the Tween compounds, in contrast, appeared to result from the unesterified fatty acids which contaminate the commercial preparations. Polyol (60), the sorbitan polyoxyethylene derivative of Tween 60 and the basic structural unit of all the Tween-type compounds, and a Tween 80 preparation which was purified by extraction of the unesterified oleic acid, were not inhibitory. Moreover, the amount of free oleic acid present as a contaminant of Tween 80 was found to be sufficient to cause significant growth inhibition. These results and the observation that E. coli does not appear to hydrolyze the esterified fatty acid of Tween 80 led to the conclusion that growth inhibition obtained with various Tween compounds probaby is a function of their respective fatty acid contaminants.

  5. Adhesive Escherichia coli in inflammatory bowel disease and infective diarrhoea.

    PubMed Central

    Burke, D. A.; Axon, A. T.

    1988-01-01

    The clinical features of ulcerative colitis and Crohn's disease are similar to those of infections of the bowel, although their cause is uncertain. Many bacteria that cause intestinal diseases adhere to the gut mucosa, and adhesion of pathogenic Escherichia coli is resistant to D-mannose. The adhesive properties of isolates of E coli were assessed by assay of adhesion to buccal epithelial cells with mannose added. The isolates were obtained from patients with inflammatory bowel diseases (50 with a relapse of ulcerative colitis, nine with ulcerative colitis in remission, 13 with Crohn's disease, and 11 with infectious diarrhoea not due to E coli) and 22 controls. The median index of adhesion to buccal epithelial cells (the proportion of cells with more than 50 adherent bacteria) for E coli from patients with ulcerative colitis in relapse was significantly higher (43%) than that for controls (5%) and patients with infectious diarrhoea (14%). The index was not significantly different among isolates from patients with ulcerative colitis in relapse, Crohn's disease (53%), and ulcerative colitis in remission (30%). If an index of adhesion of greater than 25% is taken as indicating an adhesive strain 86% of isolates of E coli from patients with inflammatory bowel disease were adhesive compared with 27% from patients with infective diarrhoea and none from controls. The adhesive properties of the isolates from patients with inflammatory bowel disease were similar to those of pathogenic intestinal E coli, raising the possibility that they may have a role in the pathogenesis of the condition; the smaller proportion of adhesive isolates in patients with infective diarrhoea due to other bacteria suggests that the organism may be of primary importance rather than arising secondarily. Images a PMID:3044496

  6. Pathogenic Escherichia coli in rural household container waters.

    PubMed

    Jagals, P; Barnard, T G; Mokoena, M M; Ashbolt, N; Roser, D J

    2013-01-01

    Plastic containers in the range of 5-20 L are widely used - especially in rural African settings - to collect, transport and store water for domestic use, including drinking, bathing and hygiene. The pathogen content of the waters in these containers has not been adequately characterized as yet. This paper presents the primary findings of a synoptic survey of drinking water quality samples from these containers and involved collection of bacterial indicator and pathogenicity gene data. In total, 571 samples of a variety of waters were taken in rural communities in South Africa and the Escherichia coli numbers measured. Of the E. coli positive samples, 46% (n = 148) were screened for the presence of E. coli pathogen gene markers. Though synoptic, the survey provided many insights into the issues that drove the study. Container use markedly degraded water quality as judged by indicator counts, even where improved water supply services were in place. Household container use also appeared to promote regrowth or contamination of containers with pathogenic E. coli strains. Polymerase chain reaction (PCR) analysis also showed that the diversity of potential pathogenic E. coli carrying virulence genes was great. All seven genes screened for (Ial, Stx1, Stx2, EaeA, Eagg, ST, LT) were found in the waters, alone or as mixtures (number of different combinations = 31) including those characteristic of the more dangerous invasive and haemorrhagic E. coli strains. Given the central role of containers in the management of water supply to rural communities, it is clear the microbiology of these waters requires much further characterization.

  7. Dynamic regulation of extracellular ATP in Escherichia coli.

    PubMed

    Alvarez, Cora Lilia; Corradi, Gerardo; Lauri, Natalia; Marginedas-Freixa, Irene; Leal Denis, María Florencia; Enrique, Nicolás; Mate, Sabina María; Milesi, Verónica; Ostuni, Mariano Anibal; Herlax, Vanesa; Schwarzbaum, Pablo Julio

    2017-04-04

    We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [(32)P]Pi released from [γ-(32)P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-(32)P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

  8. Expression of a synthetic pertussis toxin operon in Escherichia coli.

    PubMed

    Pozza, T D; Yan, H; Walker, M J

    1997-06-01

    Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A/B bacterial toxin consisting of five subunits S1-S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli beta-lactamase leader sequence. We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

  9. A structural view of the dissociation of Escherichia coli tryptophanase.

    PubMed

    Green, Keren; Qasim, Nasrin; Gdaelvsky, Garik; Kogan, Anna; Goldgur, Yehuda; Parola, Abraham H; Lotan, Ofra; Almog, Orna

    2015-12-01

    Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.

  10. Genomic analysis of extra-intestinal pathogenic Escherichia coli urosepsis.

    PubMed

    McNally, A; Alhashash, F; Collins, M; Alqasim, A; Paszckiewicz, K; Weston, V; Diggle, M

    2013-08-01

    Urosepsis is a bacteraemia infection caused by an organism previously causing an infection in the urinary tract of a patient, a diagnosis which has been classically confirmed by culture of the same species of bacteria from both blood and urine samples. Given the new insights afforded by sequencing technologies into the complicated population structures of infectious agents affecting humans, we sought to investigate urosepsis by comparing the genome sequences of blood and urine isolates of Escherichia coli from five patients with urosepsis. The results confirm the classical urosepsis hypothesis in four of the five cases, but also show the complex nature of extra-intestinal E. coli infection in the fifth case, where three distinct strains caused two distinct infections. Additionally, we show there is little to no variation in the bacterial genome as it progressed from urine to blood, and also present a minimal set of virulence genes required for bacteraemia in E. coli based on gene association. These suggest that most E. coli have the genetic propensity to cause bacteraemia.

  11. Characterization of a second lysine decarboxylase isolated from Escherichia coli.

    PubMed Central

    Kikuchi, Y; Kojima, H; Tanaka, T; Takatsuka, Y; Kamio, Y

    1997-01-01

    We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli. PMID:9226257

  12. A second DNA methyltransferase repair enzyme in Escherichia coli.

    PubMed Central

    Rebeck, G W; Coons, S; Carroll, P; Samson, L

    1988-01-01

    The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains. Images PMID:3283737

  13. Redefining the requisite lipopolysaccharide structure in Escherichia coli.

    PubMed

    Meredith, Timothy C; Aggarwal, Parag; Mamat, Uwe; Lindner, Buko; Woodard, Ronald W

    2006-02-17

    Gram-negative bacteria possess an asymmetric lipid bilayer surrounding the cell wall, the outer membrane (OM). The OM inner leaflet is primarily composed of various glycerophospholipids, whereas the outer leaflet predominantly contains the unique amphiphilic macromolecule, lipopolysaccharide (LPS or endotoxin). The majority of all gram-negative bacteria elaborate LPS containing at least one 2-keto 3-deoxy-D-manno-octulosonate (Kdo) molecule. The minimal LPS structure required for growth of Escherichia coli has long been recognized as two Kdo residues attached to lipid A, inextricably linking viability to toxicity. Here we report the construction and characterization of the nonconditional E. coli K-12 suppressor strain KPM22 that lacks Kdo and is viable despite predominantly elaborating the endotoxically inactive LPS precursor lipid IV(A). Our results challenge the established E. coli Kdo2-lipid A dogma, indicating that the previously observed and well-documented dependence of cell viability on the synthesis of Kdo stems from a lethal pleiotropy precipitated after the depletion of the carbohydrate, rather than an inherent need for the Kdo molecule itself as an indispensable structural component of the OM LPS layer. Inclusion of the inner membrane LPS transporter MsbA on a multicopy plasmid partially suppresses the lethal deltaKdo phenotype directly in the auxotrophic parent strain, suggesting increased rates of nonglycosylated lipid A transport can, in part, compensate for Kdo depletion. The unprecedented nature of a lipid IV(A) OM redefines the requisite LPS structure for viability in E. coli.

  14. Resistance patterns of Escherichia coli causing urinary tract infection

    PubMed Central

    Ferdosi-Shahandashti, Elaheh; Javanian, Mostafa; Moradian-Kouchaksaraei, Masoomeh; Yeganeh, Babak; Bijani, Ali; Motevaseli, Elahe; Moradian- Kouchaksaraei, Fatemeh

    2015-01-01

    Background: Urinary tract infection (UTI) is one of the most prevalent infectious diseases and Escherichia coli is its common cause. The aim of this study was to assess the resistance patterns of E.coli in urinary tract infections and to determine the susceptibility of E.coli to commonly used antimicrobials and also to evaluate the options for empirical treatment of UTI. Methods: This study was conducted in the Ayatollah Rouhani Teaching Hospital of Babol Medical Sciences University in North of Iran. Between January of 2013 to December 2013, antimicrobial susceptibility tests were done by disk diffusion and microdilution method. Growth of >=105 cfu/ml was considered as positive urine test. Ten commonly used antibiotics were examined for susceptibility test. Data and the results were collected and analyzed. Results: E.coli grew in 57 urine samples. Imipenem, ofloxacin, ciprofloxacin were the most sensitive antibiotics at 87.7%, 87.7% and 78.9% respectively. Whereas, cotrimoxazole, cefexime, cefotaxcime and ceftriaxone were the most resistant antibiotics. Antibiotic sensitivity of disk diffusion compared minimum inhibitory concentration (MIC) detected by microdilution had the sensitivity, specificity, positive predictive value and negative predictive value of 82%, 98%, 99% and 74%, respectively. Conclusion: Imipenem, ofloxacin and ciprofloxacin should be used in empirical therapy of UTI. PMID:26644881

  15. Curli fimbria: an Escherichia coli adhesin associated with human cystitis.

    PubMed

    Cordeiro, Melina Aparecida; Werle, Catierine Hirsch; Milanez, Guilherme Paier; Yano, Tomomasa

    2016-01-01

    Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-d-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.

  16. Magnetically-Actuated Escherichia coli System for Micro Lithography

    NASA Astrophysics Data System (ADS)

    Lauback, S.; Brown, E.; Pérez-Guzman, L.; Peace, C.; Pierce, C.; Lower, B. H.; Lower, S. K.; Sooryakumar, R.

    2015-03-01

    Technologies that control matter at the nano- and micro-scale are crucial for developing new engineered materials and devices. While the more traditional approaches for such manipulations often depend on lithographic fabrication, they can be expanded upon by taking advantage of the biological systems within a living cell which also operate on the nano- and micro- scale. In this study, a system is being developed to functionalize a targeted location on the surface of a chip with the protein AmCyan from transformed Escherichia coli cells. Using established methods in molecular biology where a plasmid with the amcyan gene sequence is inserted into the cell, E. coli are engineered to express the AmCyan protein on their outer surface. In order to transport the cells to the targeted location, the transformed E. coli are labeled with superparamagnetic micro-beads which exert directed forces on the cells in an external field. Preliminary results of the protein expression on E. coli, the transport of the cell through weak magnetic fields to targeted locations and the potential to transfer protein from the cell to the chip surface will be presented.

  17. Production of 3-O-xylosyl quercetin in Escherichia coli.

    PubMed

    Pandey, Ramesh Prasad; Malla, Sailesh; Simkhada, Dinesh; Kim, Byung-Gee; Sohng, Jae Kyung

    2013-03-01

    Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/∆pgi, E. coli BL21(DE3)/∆zwf, E. coli BL21(DE3)/∆pgi∆zwf, and E. coli BL21(DE3)/∆pgi∆zwf∆ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/∆pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/∆pgi∆zwf∆ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.

  18. Extraintestinal Escherichia coli carrying virulence genes in coastal marine sediments.

    PubMed

    Luna, G M; Vignaroli, C; Rinaldi, C; Pusceddu, A; Nicoletti, L; Gabellini, M; Danovaro, R; Biavasco, F

    2010-09-01

    Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx(1), stx(2), aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological

  19. Biosurfactins production by Bacillus amyloliquefaciens R3 and their antibacterial activity against multi-drug resistant pathogenic E. coli.

    PubMed

    Chi, Zhe; Rong, Yan-Jun; Li, Yang; Tang, Mei-Juan; Chi, Zhen-Ming

    2015-05-01

    In this work, the anti-Escherichia coli activity of the bioactive substances produced by Bacillus amyloliquefaciens R3 was examined. A new and cheap medium for production of the anti-E. coli substances which contained 20.0 g L(-1) soybean powder, 20.0 g L(-1) wheat flour, pH 6.0 was developed. A crude surfactant concentration of 0.48 mg mL(-1) was obtained after 27 h of 10-L fermentation, and the diameter of the clear zone on the plate seeded with the pathogenic E. coli 2# was 23.3 mm. A preliminary characterization suggested that the anti-E. coli substances produced by B. amyloliquefaciens R3 were the biosurfactins (F1, F2, F3, F4, and F5) with amino acids (GLLVDLL) and hydroxy fatty acids (of 12-15 carbons in length). It was found that all the strains of the pathogenic E. coli showed resistance to several different antibiotics, suggesting that they were the multi-drug resistance and all the strains of the pathogenic E. coli were sensitive to the biosurfactins, indicating that the biosurfactins produced by B. amyloliquefaciens R3 had a broad spectrum of antibacterial activity against the pathogenic E. coli with multi-drug resistant profiles. After the treatment with the purified biosurfactin (F1), the cell membrane of both the whole cells and protoplasts of the E. coli 2# was damaged and the whole cells of the bacterium were broken.

  20. Enteroaggregative Escherichia coli an emergent pathogen with different virulence properties.

    PubMed

    Villaseca, J M; Hernández, U; Sainz-Espuñes, T R; Rosario, C; Eslava, C

    2005-01-01

    Enteroaggregative Escherichia coli (EAEC) is an emergent bacterial pathogen. The first studies in developing countries with EAEC strains, showed that this bacterium was associated with persistent diarrhea. However, new studies showed that EAEC may be associated also with acute diarrhea, with both nosocomial and community outbreaks worldwide, and as an important pathogen of diarrheal disease in human immunodeficiency virus-infected adults. EAEC strains are recognized by their characteristic aggregative adherence or "stacked-brick" pattern to epithelial cells. Although the pathogenesis of EAEC infection is not well understood, cellular changes observed in animal models and in vitro assays, suggested that the alterations in the intestinal mucosa during EAEC infection are associated with adherence factors and toxins production. The damage has been associated with the release of inflammatory mediators, which may contribute also to the intestinal illness. The dissemination of the high pathogenicity island from Yersinia pestis evolutionary group to EAEC has been show; different studies suggest that it may contribute to the virulence of EAEC strains. Molecular methods to investigate the presence of plasmid and chromosomal EAEC-associated virulence markers, have been used for the characterization and epidemiological studies of EAEC strains. Although the clinical and epidemiological importance of EAEC have been demonstrated in different studies, Escherichia coli strains with adherent agreggative phenotype are commonly isolated from healthy children and environmental sources. This support the necessity to study virulence factors no related with the cells adherence pattern, that show the specific EAEC pathogenic clones associated whit intestinal disease.

  1. Protein turnover in the cell cycle of Escherichia coli.

    PubMed

    Nishi, A; Kogoma, T

    1965-10-01

    Nishi, Arasuke (University of Tokyo, Tokyo, Japan), and Tokio Kogoma. Protein turnover in the cell cycle of Escherichia coli. J. Bacteriol. 90:884-890. 1965.-Protein metabolism and enzyme formation throughout the cell cycle were investigated in synchronized cultures of Escherichia coli. The cells showed a temporary cessation of the net increase of bulk protein and of constitutive beta-galactosidase activity during the division period. By contrast, when tested by short-term experiments performed with cells at different growth stages, the bacteria displayed a constant incorporation of labeled protein precursors into the protein fraction, even during the fission period. Similar results were obtained with respect to the capacities for induced enzyme formation. On the other hand, when the cells were previously labeled and then subjected to synchronization in a nonradioactive medium, the radioactivity of the protein fraction decreased temporarily by nearly 10% during the fission period and then regained its previous level at the beginning of the ensuing phase of growth. This indicates that the products of partial degradation of protein were again utilized for protein synthesis in the next cell cycle. It was concluded that the temporary lagging of net increase of bulk protein may be due to the partial breakdown of protein occurring during the fission period.

  2. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI I.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. I. Effect of tryptophan and related compounds. J. Bacteriol. 84:979–987. 1962.—The effect of tryptophan and related compounds on tryptophanase and tryptophan synthetase formation in Escherichia coli was determined. Several of these compounds stimulated the formation of tryptophanase while concomitantly decreasing the production of synthetase. A number of tryptophan analogues were found to inhibit growth. The possible mode of action of these substances was examined further. 5-Hydroxytryptophan greatly inhibited the formation of synthetase and also reduced growth. Its inhibitory action on growth was attributed, at least partially, to the false feedback inhibition of anthranilic acid formation. Tryptamine was found to be a potent inhibitor of the activity of synthetase, as well as of the enzyme(s) involved in the synthesis of anthranilic acid from shikimic acid. However, growth reduction was only partially reversed by tryptophan. Indole-3-acetic acid and indole-3-propionic acid decreased growth and increased the formation of synthetase six- to eightfold. The action of these compounds was ascribed to their ability to block the endogenous formation of tryptophan. PMID:13959621

  3. Translocation and thermal inactivation of Shiga-toxin producing Escherichia coli in non-intact beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We compared translocation of genetically-marked strains of serotype O157:H7 Escherichia coli (ECOH) to non-O157:H7 Shiga-Toxin producing Escherichia coli (STEC) following blade tenderization of beef subprimals and the subsequent lethality of these pathogens following cooking of steaks prepared from ...

  4. F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens.

    PubMed Central

    Mergeay, M; Gerits, J

    1978-01-01

    Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4). PMID:97267

  5. Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli

    PubMed Central

    Blum, Shlomo E.; Heller, Elimelech D.; Sela, Shlomo; Elad, Daniel; Edery, Nir; Leitner, Gabriel

    2015-01-01

    Escherichia coli is a major etiological agent of intra-mammary infections (IMI) in cows, leading to acute mastitis and causing great economic losses in dairy production worldwide. Particular strains cause persistent IMI, leading to recurrent mastitis. Virulence factors of mammary pathogenic E. coli (MPEC) involved pathogenesis of mastitis as well as those differentiating strains causing acute or persistent mastitis are largely unknown. This study aimed to identify virulence markers in MPEC through whole genome and phenome comparative analysis. MPEC strains causing acute (VL2874 and P4) or persistent (VL2732) mastitis were compared to an environmental strain (K71) and to the genomes of strains representing different E. coli pathotypes. Intra-mammary challenge in mice confirmed experimentally that the strains studied here have different pathogenic potential, and that the environmental strain K71 is non-pathogenic in the mammary gland. Analysis of whole genome sequences and predicted proteomes revealed high similarity among MPEC, whereas MPEC significantly differed from the non-mammary pathogenic strain K71, and from E. coli genomes from other pathotypes. Functional features identified in MPEC genomes and lacking in the non-mammary pathogenic strain were associated with synthesis of lipopolysaccharide and other membrane antigens, ferric-dicitrate iron acquisition and sugars metabolism. Features associated with cytotoxicity or intra-cellular survival were found specifically in the genomes of strains from severe and acute (VL2874) or persistent (VL2732) mastitis, respectively. MPEC genomes were relatively similar to strain K-12, which was subsequently shown here to be possibly pathogenic in the mammary gland. Phenome analysis showed that the persistent MPEC was the most versatile in terms of nutrients metabolized and acute MPEC the least. Among phenotypes unique to MPEC compared to the non-mammary pathogenic strain were uric acid and D-serine metabolism. This study

  6. Nucleotide sequence of an Escherichia coli chromosomal hemolysin.

    PubMed Central

    Felmlee, T; Pellett, S; Welch, R A

    1985-01-01

    We determined the DNA sequence of an 8,211-base-pair region encompassing the chromosomal hemolysin, molecularly cloned from an O4 serotype strain of Escherichia coli. All four hemolysin cistrons (transcriptional order, C, A, B, and D) were encoded on the same DNA strand, and their predicted molecular masses were, respectively, 19.7, 109.8, 79.9, and 54.6 kilodaltons. The identification of pSF4000-encoded polypeptides in E. coli minicells corroborated the assignment of the predicted polypeptides for hlyC, hlyA, and hlyD. However, based on the minicell results, two polypeptides appeared to be encoded on the hlyB region, one similar in size to the predicted molecular mass of 79.9 kilodaltons, and the other a smaller 46-kilodalton polypeptide. The four hemolysin gene displayed similar codon usage, which is atypical for E. coli. This reflects the low guanine-plus-cytosine content (40.2%) of the hemolysin DNA sequence and suggests the non-E. coli origin of the hemolysin determinant. In vitro-derived deletions of the hemolysin recombinant plasmid pSF4000 indicated that a region between 433 and 301 base pairs upstream of the putative start of hlyC is necessary for hemolysin synthesis. Based on the DNA sequence, a stem-loop transcription terminator-like structure (a 16-base-pair stem followed by seven uridylates) in the mRNA was predicted distal to the C-terminal end of hlyA. A model for the general transcriptional organization of the E. coli hemolysin determinant is presented. Images PMID:3891743

  7. Discovery of Escherichia coli CRISPR sequences in an undergraduate laboratory.

    PubMed

    Militello, Kevin T; Lazatin, Justine C

    2016-09-28

    Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some similarity to RNA interference, and the ability to utilize reconstituted CRISPR systems for genome editing in numerous organisms. In order to introduce CRISPR biology into an undergraduate upper-level laboratory, a five-week set of exercises was designed to allow students to examine the CRISPR status of uncharacterized Escherichia coli strains and to allow the discovery of new repeats and spacers. Students started the project by isolating genomic DNA from E. coli and amplifying the iap CRISPR locus using the polymerase chain reaction (PCR). The PCR products were analyzed by Sanger DNA sequencing, and the sequences were examined for the presence of CRISPR repeat sequences. The regions between the repeats, the spacers, were extracted and analyzed with BLASTN searches. Overall, CRISPR loci were sequenced from several previously uncharacterized E. coli strains and one E. coli K-12 strain. Sanger DNA sequencing resulted in the discovery of 36 spacer sequences and their corresponding surrounding repeat sequences. Five of the spacers were homologous to foreign (non-E. coli) DNA. Assessment of the laboratory indicates that improvements were made in the ability of students to answer questions relating to the structure and function of CRISPRs. Future directions of the laboratory are presented and discussed. © 2016 by The International Union of Biochemistry and Molecular Biology, 2016.

  8. Anaerobic respiration of Escherichia coli in the mouse intestine.

    PubMed

    Jones, Shari A; Gibson, Terri; Maltby, Rosalie C; Chowdhury, Fatema Z; Stewart, Valley; Cohen, Paul S; Conway, Tyrrell

    2011-10-01

    The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coli is the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coli uses to maximize its competitiveness and achieve the highest possible population in

  9. A Novel Putrescine Exporter SapBCDF of Escherichia coli.

    PubMed

    Sugiyama, Yuta; Nakamura, Atsuo; Matsumoto, Mitsuharu; Kanbe, Ayaka; Sakanaka, Mikiyasu; Higashi, Kyohei; Igarashi, Kazuei; Katayama, Takane; Suzuki, Hideyuki; Kurihara, Shin

    2016-12-16

    Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 μm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.

  10. Genetic determinants of heat resistance in Escherichia coli

    PubMed Central

    Mercer, Ryan G.; Zheng, Jinshui; Garcia-Hernandez, Rigoberto; Ruan, Lifang; Gänzle, Michael G.; McMullen, Lynn M.

    2015-01-01

    Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; or as heat sensitive. A ~14 kb genomic island containing 16 predicted open reading frames encoding putative heat shock proteins and proteases was identified only in highly heat resistant strains. The genomic island was termed the locus of heat resistance (LHR). This putative operon is flanked by mobile elements and possesses >99% sequence identity to genomic islands contributing to heat resistance in Cronobacter sakazakii and Klebsiella pneumoniae. An additional 41 LHR sequences with >87% sequence identity were identified in 11 different species of β- and γ-proteobacteria. Cloning of the full length LHR conferred high heat resistance to the heat sensitive E. coli AW1.7ΔpHR1 and DH5α. The presence of the LHR correlates perfectly to heat resistance in several species of Enterobacteriaceae and occurs at a frequency of 2% of all E. coli genomes, including pathogenic strains. This study suggests the LHR has been laterally exchanged among the β- and γ-proteobacteria and is a reliable indicator of high heat resistance in E. coli. PMID:26441869

  11. Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli.

    PubMed

    Chen, Weiwei; Yu, Hongwei; Ye, Lidan

    2016-07-01

    The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.

  12. FILAMENT FORMATION BY ESCHERICHIA COLI AT INCREASED HYDROSTATIC PRESSURES1

    PubMed Central

    Zobell, Claude E.; Cobet, Andre B.

    1964-01-01

    ZoBell, Claude E. (University of California, La Jolla), and Andre B. Cobet. Filament formation by Escherichia coli at increased hydrostatic pressures. J. Bacteriol. 87:710–719. 1964.—The reproduction as well as the growth of Escherichia coli is retarded by hydrostatic pressures ranging from 200 to 500 atm. Reproduction was indicated by an increase in the number of cells determined by plating on EMB Agar as well as by direct microscopic counts. Growth, which is not necessarily synonymous with reproduction, was indicated by increase in dry weight and protein content of the bacterial biomass. At increased pressures, cells of three different strains of E. coli tended to form long filaments. Whereas most normal cells of E. coli that developed at 1 atm were only about 2 μ long, the mean length of those that developed at 475 atm was 2.93 μ for strain R4, 3.99 μ for strain S, and 5.82 μ for strain B cells. Nearly 90% of the bacterial biomass produced at 475 atm by strain B was found in filaments exceeding 5 μ in length; 74.7 and 16.4% of the biomass produced at 475 atm by strains S and R4, respectively, occurred in such filaments. Strain R4 formed fewer and shorter (5 to 35 μ) filaments than did the other two strains, whose filaments ranged in length from 5 to >100 μ. The bacterial biomass produced at all pressures had approximately the same content of protein and nucleic acids. But at increased pressures appreciably more ribonucleic acid (RNA) and proportionately less deoxyribonucleic acid (DNA) was found per unit of biomass. Whereas the RNA content per cell increased with cell length, the amount of DNA was nearly the same in long filaments formed at increased pressure as in cells of normal length formed at 1 atm. The inverse relationship between the concentration of DNA and cell length in all three strains of E. coli suggests that the failure of DNA to replicate at increased pressure may be responsible for a repression of cell division and consequent filament

  13. Metabolic self-organization of bioluminescent Escherichia coli.

    PubMed

    Simkus, Remigijus; Baronas, Romas

    2011-01-01

    A possible reason for the complexity of the signals produced by bioluminescent biosensors might be self-organization of the cells. In order to verify this possibility, bioluminescence images of cultures of lux gene reporter Escherichia coli were recorded for several hours after being placed into 8-10 mm diameter cylindrical containers. It was found that luminous cells distribute near the three-phase contact line, forming irregular azimuthal waves. As we show, space-time plots of quasi-one-dimensional bioluminescence measured along the contact line can be simulated by reaction-diffusion-chemotaxis equations, in which the reaction term for the cells is a logistic (autocatalytic) growth function. It was found that the growth rate of the luminous cells (~0.02 s(-1)) is >100 times higher than the growth rate of E. coli. We provide an explanation for this result by assuming that E. coli exhibits considerable respiratory flexibility (the ability of oxygen-induced switching from one metabolic pathway to another). According to the simple two-state model presented here, the number of oxic (luminous) cells grows at the expense of anoxic (dark) cells, whereas the total number of (oxic and anoxic) cells remains unchanged. It is conjectured that the corresponding reaction-diffusion-chemotaxis model for bioluminescence pattern formation can be considered as a model for the energy-taxis and metabolic self-organization in the population of the metabolically flexible bacteria under hypoxic conditions.

  14. Properties of a Clostridium thermocellum Endoglucanase Produced in Escherichia coli

    PubMed Central

    Schwarz, Wolfgang H.; Gräbnitz, Folke; Staudenbauer, Walter L.

    1986-01-01

    A cellulase gene of Clostridium thermocellum was transferred to Escherichia coli by molecular cloning with bacteriophage lambda and plasmid vectors and shown to be indentical with the celA gene. The celA gene product was purified from extracts of plasmid-bearing E. coli cells by heat treatment and chromatography on DEAE-Trisacryl. It was characterized as a thermophilic endo-β-1,4-glucanase, the properties of which closely resemble those of endoglucanase A previously isolated from C. thermocellum supernatants. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme purified from E. coli exhibited two protein bands with molecular weights of 49,000 and 52,000. It had a temperature optimum at 75°C and was stable for several hours at 60°C. Endoglucanase activity was optimal between pH 5.5 and 6.5. The enzyme was insensitive against end product inhibition by glucose and cellobiose and remarkably resistant to the denaturing effects of detergents and organic solvents. It was capable of degrading, in addition to cellulosic substrates, glucans with alternating β-1,4 and β-1,3 linkages such as barley β-glucan and lichenan. PMID:16347088

  15. Comprehensive Mapping of the Escherichia coli Flagellar Regulatory Network

    PubMed Central

    Fitzgerald, Devon M.; Bonocora, Richard P.; Wade, Joseph T.

    2014-01-01

    Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process. PMID:25275371

  16. Global regulation of gene expression in Escherichia coli.

    PubMed Central

    Chuang, S E; Daniels, D L; Blattner, F R

    1993-01-01

    Global transcription responses of Escherichia coli to various stimuli or genetic defects were studied by measuring mRNA levels in about 400 segments of the genome. Measuring mRNA levels was done by analyzing hybridization to DNA dot blots made with overlapping lambda clones spanning the genome of E. coli K-12. Conditions examined included isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, heat shock, osmotic shock, starvation for various nutrients, entrance of cells into the stationary phase of growth, anaerobic growth in a tube, growth in the gnotobiotic mouse gut, and effects of pleiotropic mutations rpoH, himA, topA, and crp. Most mapped genes known to be regulated by a particular situation were successfully detected. In addition, many chromosomal regions containing no previously known regulated genes were discovered that responded to various stimuli. This new method for studying globally regulated genetic systems in E. coli combines detection, cloning, and physical mapping of a battery of coregulated genes in one step. Images PMID:8458845

  17. Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli

    PubMed Central

    Garrett, Teresa A.; Raetz, Christian R. H.; Richardson, Travis; Kordestani, Reza; Son, Jennifer D.; Rose, Rebecca L.

    2009-01-01

    Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655. PMID:19096047

  18. Improving alkane synthesis in Escherichia coli via metabolic engineering.

    PubMed

    Song, Xuejiao; Yu, Haiying; Zhu, Kun

    2016-01-01

    Concerns about energy security and global petroleum supply have made the production of renewable biofuels an industrial imperative. The ideal biofuels are n-alkanes in that they are chemically and structurally identical to the fossil fuels and can "drop in" to the transportation infrastructure. In this work, an Escherichia coli strain that produces n-alkanes was constructed by heterologous expression of acyl-acyl carrier protein (ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) from Synechococcus elongatus PCC7942. The accumulation of alkanes ranged from 3.1 to 24.0 mg/L using different expressing strategies. Deletion of yqhD, an inherent aldehyde reductase in E. coli, or overexpression of fadR, an activator for fatty acid biosynthesis, exhibited a nearly twofold increase in alkane titers, respectively. Combining yqhD deletion and fadR overexpression resulted in a production titer of 255.6 mg/L in E. coli, and heptadecene was the most abundant product.

  19. Production of 2-methyl-1-butanol in engineered Escherichia coli.

    PubMed

    Cann, Anthony F; Liao, James C

    2008-11-01

    Recent progress has been made in the production of higher alcohols by harnessing the power of natural amino acid biosynthetic pathways. Here, we describe the first strain of Escherichia coli developed to produce the higher alcohol and potential new biofuel 2-methyl-1-butanol (2MB). To accomplish this, we explored the biodiversity of enzymes catalyzing key parts of the isoleucine biosynthetic pathway, finding that AHAS II (ilvGM) from Salmonella typhimurium and threonine deaminase (ilvA) from Corynebacterium glutamicum improve 2MB production the most. Overexpression of the native threonine biosynthetic operon (thrABC) on plasmid without the native transcription regulation also improved 2MB production in E. coli. Finally, we knocked out competing pathways upstream of threonine production (DeltametA, Deltatdh) to increase its availability for further improvement of 2MB production. This work led to a strain of E. coli that produces 1.25 g/L 2MB in 24 h, a total alcohol content of 3 g/L, and with yields of up to 0.17 g 2MB/g glucose.

  20. Reductive transformation of TNT by Escherichia coli: pathway description.

    PubMed

    Yin, Hong; Wood, Thomas K; Smets, Barth F

    2005-05-01

    The reductive transformation of 2,4,6-trinitrotoluene (TNT) was studied using aerobically grown Escherichia coli cultures. In the absence of an external carbon or energy source, E. coli resting cells transformed TNT to hydroxylaminodinitrotoluenes (2HADNT, 4HADNT, with 4HADNT as the dominant isomer), aminodinitrotoluenes (4ADNT, with sporadic detection of 2ADNT), 2,4-di(hydroxylamino)-6-nitrotoluene (24D(HA)6NT), 2,4-diamino-6-nitrotoluene (24DA6NT), and an additional compound which was tentatively identified as a (hydroxylamino)aminonitrotoluene isomer via gas chromatography/mass spectroscopy and spectral analysis. The resting cell assay, performed in an oxygen-free atmosphere, avoided formation of azoxy dimers and provided good mass balances. Significant preference for reduction in the para versus ortho position was detected. The formation of 24D(HA)6NT, but not ADNT, appeared inhibited by the presence of TNT. The rate and extent of TNT reduction were significantly enhanced at higher cell densities, or by supplying an exogenous reducing power source, revealing the importance of enzyme concentration and reducing power. Whether the oxygen-insensitive E. coli nitroreductases, encoded by nfsA and nfsB, directly catalyze the TNT reduction or account for the complete TNT transformation pathway, remains to be determined.

  1. Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase.

    PubMed

    Romão, Célia V; Vicente, João B; Borges, Patrícia T; Victor, Bruno L; Lamosa, Pedro; Silva, Elísio; Pereira, Luís; Bandeiras, Tiago M; Soares, Cláudio M; Carrondo, Maria A; Turner, David; Teixeira, Miguel; Frazão, Carlos

    2016-11-20

    Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O2 and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species. E. coli FlRd has a clear preference for NO as a substrate in vitro, exhibiting a very low reactivity toward O2. To contribute to the understanding of the structural features defining this substrate selectivity, we determined the crystallographic structure of E. coli FlRd, both in the isolated and reduced states. The overall tetrameric structure revealed a highly conserved flavodiiron core domain, with a metallo-β-lactamase-like domain containing a diiron center, and a flavodoxin domain with a flavin mononucleotide cofactor. The metal center in the oxidized state has a μ-hydroxo bridge coordinating the two irons, while in the reduced state, this moiety is not detected. Since only the flavodiiron domain was observed in these crystal structures, the structure of the rubredoxin domain was determined by NMR. Tunnels for the substrates were identified, and through molecular dynamics simulations, no differences for O2 or NO permeation were found. The present data represent the first structure for a NO-selective FDP.

  2. Identification of diarrheagenic Escherichia coli strains from avian organic fertilizers.

    PubMed

    Puño-Sarmiento, Juan; Gazal, Luis Eduardo; Medeiros, Leonardo P; Nishio, Erick K; Kobayashi, Renata K T; Nakazato, Gerson

    2014-08-28

    The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC) strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%), three strains as Shiga toxin-producing (STEC; 4.7%), 10 strains as enteroaggregative (EAEC; 12.5%), but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections.

  3. Heterologous biosynthesis and manipulation of alkanes in Escherichia coli.

    PubMed

    Cao, Ying-Xiu; Xiao, Wen-Hai; Zhang, Jin-Lai; Xie, Ze-Xiong; Ding, Ming-Zhu; Yuan, Ying-Jin

    2016-11-01

    Biosynthesis of alkanes in microbial foundries offers a sustainable and green supplement to traditional fossil fuels. The dynamic equilibrium of fatty aldehydes, key intermediates, played a critical role in microbial alkanes production, due to the poor catalytic capability of aldehyde deformylating oxygenase (ADO). In our study, exploration of competitive pathway together with multi-modular optimization was utilized to improve fatty aldehydes balance and consequently enhance alkanes formation in Escherichia coli. Endogenous fatty alcohol formation was supposed to be competitive with alkane production, since both of the two routes consumed the same intermediate-fatty aldehyde. Nevertheless, in our case, alkanes production in E. coli was enhanced from trace amount to 58.8mg/L by the facilitation of moderate fatty alcohol biosynthesis, which was validated by deletion of endogenous aldehyde reductase (AHR), overexpression of fatty alcohol oxidase (FAO) and consequent transcriptional assay of aar, ado and adhP genes. Moreover, alkanes production was further improved to 81.8mg/L, 86.6mg/L or 101.7mg/L by manipulation of fatty acid biosynthesis, lipids degradation or electron transfer system modules, which directly referenced to fatty aldehydes dynamic pools. A titer of 1.31g/L alkanes was achieved in 2.5L fed-batch fermentation, which was the highest reported titer in E. coli. Our research has offered a reference for chemical overproduction in microbial cell factories facilitated by exploring competitive pathway.

  4. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli

    PubMed Central

    Kay, Emily J.; Yates, Laura E.; Terra, Vanessa S.; Cuccui, Jon; Wren, Brendan W.

    2016-01-01

    Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology. PMID:27110302

  5. Characterization of the YdeO regulon in Escherichia coli.

    PubMed

    Yamanaka, Yuki; Oshima, Taku; Ishihama, Akira; Yamamoto, Kaneyoshi

    2014-01-01

    Enterobacteria are able to survive under stressful conditions within animals, such as acidic conditions in the stomach, bile salts during transfer to the intestine and anaerobic conditions within the intestine. The glutamate-dependent (GAD) system plays a major role in acid resistance in Escherichia coli, and expression of the GAD system is controlled by the regulatory cascade consisting of EvgAS > YdeO > GadE. To understand the YdeO regulon in vivo, we used ChIP-chip to interrogate the E. coli genome for candidate YdeO binding sites. All of the seven operons identified by ChIP-chip as being potentially regulated by YdeO were confirmed as being under the direct control of YdeO using RT-qPCR, EMSA, DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified four stress-response transcription factors, DctR, NhaR, GadE, and GadW and enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation of the GAD system and NhaR is an activator for the sodium/proton antiporter gene. In conjunction with co-transcribed Slp, DctR is involved in protection against metabolic endoproducts under acidic conditions. Taken all together, we suggest that YdeO is a key regulator of E. coli survival in both acidic and anaerobic conditions.

  6. Baicalin Protects Mice from Lethal Infection by Enterohemorrhagic Escherichia coli.

    PubMed

    Zhang, Yong; Qi, Zhimin; Liu, Yan; He, Wenqi; Yang, Cheng; Wang, Quan; Dong, Jing; Deng, Xuming

    2017-01-01

    Shiga-like toxin-producing Escherichia coli (STEC) O157:H7 poses grave challenges to public health by its ability to cause severe colonic diseases and renal failure in both human and animals. Shiga-like toxins are the major pathogenic factor for some highly virulent E. coli expecially Shiga-like toxin 2. Conventional treatments such as antibiotics can facilitate the release of the toxin thus potentially exacerbate the diseases. Small molecule inhibitors and antibodies capable of neutralizing the toxins are the two major venues for the development of therapeutics against enterohemorrhagic serotype E. coli infection. While promising and potentially effective at clinical settings, these approaches need to overcome obstacles such as the limited routes of administration, responses from the host immune system, which are known to differ greatly among individuals. Our previous studies demonstrate that Baicalin (BAI), a flavonoid compound isolated from Scutellaria baicalensis protects against rStx2-induced cell cytotoxicity and also protects mice from lethal rStx2 challenges by inducing Stx2 to form inactive oligomers. In this manuscript, we present some exciting work showing that baicalin is an effective agent for therapeutic treatment of STEC O157:H7 infection.

  7. Microaerobic conversion of glycerol to ethanol in Escherichia coli.

    PubMed

    Wong, Matthew S; Li, Mai; Black, Ryan W; Le, Thao Q; Puthli, Sharon; Campbell, Paul; Monticello, Daniel J

    2014-05-01

    Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process.

  8. Escherichia coli Chromosomal Loci Segregate from Midcell with Universal Dynamics.

    PubMed

    Cass, Julie A; Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2016-06-21

    The structure of the Escherichia coli chromosome is inherently dynamic over the duration of the cell cycle. Genetic loci undergo both stochastic motion around their initial positions and directed motion to opposite poles of the rod-shaped cell during segregation. We developed a quantitative method to characterize cell-cycle dynamics of the E. coli chromosome to probe the chromosomal steady-state mobility and segregation process. By tracking fluorescently labeled chromosomal loci in thousands of cells throughout the entire cell cycle, our method allows for the statistical analysis of locus position and motion, the step-size distribution for movement during segregation, and the locus drift velocity. The robust statistics of our detailed analysis of the wild-type E. coli nucleoid allow us to observe loci moving toward midcell before segregation occurs, consistent with a replication factory model. Then, as segregation initiates, we perform a detailed characterization of the average segregation velocity of loci. Contrary to origin-centric models of segregation, which predict distinct dynamics for oriC-proximal versus oriC-distal loci, we find that the dynamics of loci were universal and independent of genetic position.

  9. The D-allose operon of Escherichia coli K-12.

    PubMed Central

    Kim, C; Song, S; Park, C

    1997-01-01

    Escherichia coli K-12 can utilize D-allose, an all-cis hexose, as a sole carbon source. The operon responsible for D-allose metabolism was localized at 92.8 min of the E. coli linkage map. It consists of six genes, alsRBACEK, which are inducible by D-allose and are under the control of the repressor gene alsR. This operon is also subject to catabolite repression. Three genes, alsB, alsA, and alsC, appear to be necessary for transport of D-allose. D-Allose-binding protein, encoded by alsB, is a periplasmic protein that has an affinity for D-allose, with a Kd of 0.33 microM. As was found for other binding-protein-mediated ABC transporters, the allose transport system includes an ATP-binding component (AlsA) and a transmembrane protein (AlsC). It was found that AlsE (a putative D-allulose-6-phosphate 3-epimerase), but not AlsK (a putative D-allose kinase), is necessary for allose metabolism. During this study, we observed that the D-allose transporter is partially responsible for the low-affinity transport of D-ribose and that strain W3110, an E. coli prototroph, has a defect in the transport of D-allose mediated by the allose permease. PMID:9401019

  10. Escherichia coli bacteria detection by using graphene-based biosensor.

    PubMed

    Akbari, Elnaz; Buntat, Zolkafle; Afroozeh, Abdolkarim; Zeinalinezhad, Alireza; Nikoukar, Ali

    2015-10-01

    Graphene is an allotrope of carbon with two-dimensional (2D) monolayer honeycombs. A larger detection area and higher sensitivity can be provided by graphene-based nanosenor because of its 2D structure. In addition, owing to its special characteristics, including electrical, optical and physical properties, graphene is known as a more suitable candidate compared to other materials used in the sensor application. A novel model employing a field-effect transistor structure using graphene is proposed and the current-voltage (I-V) characteristics of graphene are employed to model the sensing mechanism. This biosensor can detect Escherichia coli (E. coli) bacteria, providing high levels of sensitivity. It is observed that the graphene device experiences a drastic increase in conductance when exposed to E. coli bacteria at 0-10(5) cfu/ml concentration. The simple, fast response and high sensitivity of this nanoelectronic biosensor make it a suitable device in screening and functional studies of antibacterial drugs and an ideal high-throughput platform which can detect any pathogenic bacteria. Artificial neural network and support vector regression algorithms have also been used to provide other models for the I-V characteristic. A satisfactory agreement has been presented by comparison between the proposed models with the experimental data.

  11. Butyrate production under aerobic growth conditions by engineered Escherichia coli.

    PubMed

    Kataoka, Naoya; Vangnai, Alisa S; Pongtharangkul, Thunyarat; Yakushi, Toshiharu; Matsushita, Kazunobu

    2017-01-11

    Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E. coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E. coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140 mM (12.3 g/L in terms of butyric acid) after 54 h of fed-batch culture.

  12. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  13. Rotational tumbling of Escherichia coli aggregates under shear

    NASA Astrophysics Data System (ADS)

    Portela, R.; Patrício, P.; Almeida, P. L.; Sobral, R. G.; Franco, J. M.; Leal, C. R.

    2016-12-01

    Growing living cultures of Escherichia coli bacteria are investigated using real-time in situ rheology and rheoimaging measurements. In the early stages of growth (lag phase) and when subjected to a constant stationary shear, the viscosity slowly increases with the cell's population. As the bacteria reach the exponential phase of growth, the viscosity increases rapidly, with sudden and temporary abrupt decreases and recoveries. At a certain stage, corresponding grossly to the late phase of growth, when the population stabilizes, the viscosity also keeps its maximum constant value, with drops and recoveries, for a long period of time. This complex rheological behavior, which is observed to be shear strain dependent, is a consequence of two coupled effects: the cell density continuous increase and its changing interacting properties. Particular attention is given to the late phase of growth of E. coli populations under shear. Rheoimaging measurements reveal, near the static plate, a rotational motion of E. coli aggregates, collectively tumbling and flowing in the shear direction. This behavior is interpreted in the light of a simple theoretical approach based on simple rigid body mechanics.

  14. Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

    PubMed Central

    Wong, Matthew S.; Li, Mai; Black, Ryan W.; Le, Thao Q.; Puthli, Sharon; Campbell, Paul

    2014-01-01

    Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process. PMID:24584248

  15. Engineering Escherichia coli for Microbial Production of Butanone

    PubMed Central

    Srirangan, Kajan; Liu, Xuejia; Akawi, Lamees; Bruder, Mark; Moo-Young, Murray

    2016-01-01

    To expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway in Escherichia coli for heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producing E. coli host strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohologenesis under selected fermentative culture conditions were investigated. Using the engineered E. coli strain for batch cultivation with 30 g liter−1 glycerol as the carbon source, we achieved coproduction of 1.3 g liter−1 butanone and 2.9 g liter−1 acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform. PMID:26896132

  16. Baicalin Protects Mice from Lethal Infection by Enterohemorrhagic Escherichia coli

    PubMed Central

    Zhang, Yong; Qi, Zhimin; Liu, Yan; He, Wenqi; Yang, Cheng; Wang, Quan; Dong, Jing; Deng, Xuming

    2017-01-01

    Shiga-like toxin-producing Escherichia coli (STEC) O157:H7 poses grave challenges to public health by its ability to cause severe colonic diseases and renal failure in both human and animals. Shiga-like toxins are the major pathogenic factor for some highly virulent E. coli expecially Shiga-like toxin 2. Conventional treatments such as antibiotics can facilitate the release of the toxin thus potentially exacerbate the diseases. Small molecule inhibitors and antibodies capable of neutralizing the toxins are the two major venues for the development of therapeutics against enterohemorrhagic serotype E. coli infection. While promising and potentially effective at clinical settings, these approaches need to overcome obstacles such as the limited routes of administration, responses from the host immune system, which are known to differ greatly among individuals. Our previous studies demonstrate that Baicalin (BAI), a flavonoid compound isolated from Scutellaria baicalensis protects against rStx2-induced cell cytotoxicity and also protects mice from lethal rStx2 challenges by inducing Stx2 to form inactive oligomers. In this manuscript, we present some exciting work showing that baicalin is an effective agent for therapeutic treatment of STEC O157:H7 infection. PMID:28337193

  17. Characterization of Enterohemorrhagic Escherichia coli on Veal Hides and Carcasses.

    PubMed

    Bosilevac, Joseph M; Wang, Rong; Luedtke, Brandon E; Hinkley, Susanne; Wheeler, Tommy L; Koohmaraie, Mohammad

    2017-01-01

    Enterohemorrhagic Escherichia coli (EHEC) are Shiga toxin-producing E. coli associated with the most severe forms of foodborne illnesses. The U.S. Department of Agriculture, Food Safety and Inspection Service has identified a higher percentage of non-O157 EHEC compared with E. coli O157:H7-positive samples collected from veal trimmings than from products produced from other cattle slaughter classes. Therefore samples were collected from hides and preevisceration carcasses at five veal processors to assess E. coli O157:H7 and non-O157 EHEC contamination during bob veal and formula-fed veal dressing procedures. E. coli O157:H7 prevalence was measured by culture isolation and found to be on 20.3% of hides and 6.7% of carcasses. In contrast, a non-O157 EHEC molecular screening assay identified 90.3% of hides and 68.2% of carcasses as positive. Only carcass samples were taken forward to culture confirmation and 38.7% yielded one or more non-O157 EHEC isolates. The recovery of an EHEC varied by plant and sample collection date; values ranged from 2.1 to 87.8% among plants and from 4.2 to 64.2% within the same plant. Three plants were resampled after changes were made to sanitary dressing procedures. Between the two collection times at the three plants, hide-to-carcass transfer of E. coli O157:H7 and non-O157 EHEC was significantly reduced. All adulterant EHEC serogroups (O26, O45, O103, O111, O121, and O145) were isolated from veal carcasses as well as four other potentially pathogenic serogroups (O5, O84, O118, and O177). Bob veal was found to have a greater culture prevalence of E. coli O157:H7 and greater positive molecular screens for non-O157 EHEC than formula-fed veal (P < 0.05), but the percentage of culture-confirmed non-O157 EHEC was not different (P > 0.05) between the two types of calves. EHEC-O26, -O111, and -O121 were found more often in bob veal (P < 0.05), whereas EHEC-O103 was found more often in formula-fed veal (P < 0.05).

  18. Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology.

    PubMed

    Sadr, Vahideh; Saffar, Behnaz; Emamzadeh, Rahman

    2017-04-30

    Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E. coli Origami. The fusion protein with a molecular weight of approximately 35kDa was analyzed on SDS-PAGE gel. The highest expression was observed after 6h induction and the fusion protein consisted approximately 47% of the total cellular protein. The purified SUMO-hepcidin25 purity was determined to be higher than 95%, with a final yield of 3.9mgl(-)(1) of media. The recombinant hepcidin25 showed antibacterial activity against both Gram negative (Klebsiella pneumonia) and Gram positive (Staphylococcus aureus and Bacillus cereus) bacteria with minimum inhibitory concentrations (MICs) of 150μgml(-1), 18.7μg/ml(-1) and 37.5μg/ml(-1), respectively. These results indicated that thioredoxin and SUMO dual fusion system is an efficient production system for synthesis functional human hepcidin25.

  19. An external substrate-free blue/white screening system in Escherichia coli.

    PubMed

    Xie, Zhoujie; Zhang, Zhao; Cao, Zhenju; Chen, Meng; Li, Pengwei; Liu, Weifeng; Qin, Hua; Zhao, Xuejin; Tao, Yong; Chen, Yihua

    2017-03-28

    Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus. When IdgS is activated from an apo-form to a holo-form via a posttranslational modification catalyzed by Sfp, it can synthesize a blue pigment indigoidine using L-glutamine, the amino acid abundant in cells, as a substrate. The new blue/white screening system contains a recipient E. coli strain with an optimized idgS gene cassette and a cloning vector harboring an sfp gene with an in-frame insertion of a multiple cloning site close to its N-terminal. We demonstrated that the IdgS/Sfp-based blue/white screening system is a powerful alternative to the lacZα-based screening system, which does not require any external substrate addition.

  20. Involvement of pnp in survival of UV radiation in Escherichia coli K-12.

    PubMed

    Rath, Devashish; Mangoli, Suhas H; Pagedar, Amruta R; Jawali, Narendra

    2012-05-01

    Polynucleotide phosphorylase (PNPase), a multifunctional protein, is a 3'→5' exoribonuclease or exoDNase in the presence of inorganic phosphate (P(i)), and extends a 3'-OH of RNA or ssDNA in the presence of ADP or dADP. In Escherichia coli, PNPase is known to protect against H(2)O(2)- and mitomycin C-induced damage. Recent reports show that Bacillus subtilis PNPase is required for repair of H(2)O(2)-induced double-strand breaks. Here we show that absence of PNPase makes E. coli cells sensitive to UV, indicating that PNPase has a role in survival of UV radiation damage. Analyses of various DNA repair pathways show that in the absence of nucleotide excision repair, survival of UV radiation depends critically on PNPase function. Consequently, uvrA pnp, uvrB pnp and uvrC pnp strains show hypersensitivity to UV radiation. Whereas the pnp mutation is non-epistatic to recJ, recQ and recG mutations with respect to the UV-sensitivity phenotype, it is epistatic to uvrD, recB and ruvA mutations, implicating it in the recombinational repair process.

  1. An in vivo, label-free quick assay for xylose transport in Escherichia coli.

    PubMed

    Chen, Tingjian; Zhang, Jingqing; Liang, Ling; Yang, Rong; Lin, Zhanglin

    2009-07-01

    Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-beta-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20 g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms.

  2. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  3. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  4. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  5. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  6. Production of bioactive chicken follistatin315 in Escherichia coli.

    PubMed

    Lee, Sang Beum; Choi, Rocky; Park, Sung Kwon; Kim, Yong Soo

    2014-12-01

    Follistatin (FST) binds to myostatin (MSTN), a potent negative regulator of skeletal muscle growth. Inhibition of MSTN activity by FST treatment has shown to enhance muscle growth as well as ameliorate symptoms of muscular dystrophy in animal models, illustrating the potential of FST as an agent to enhance muscle growth in animal agriculture or to treat muscle wasting conditions or disease in humans. Therefore, we designed a study to produce biologically active recombinant chicken FST315 (chFST315) in an Escherichia coli host. Since FST contains multiple intramolecular disulfide bonds, we expressed chFST315 protein in either a system that utilizes a periplasmic expression strategy, or a genetically modified E. coli system (SHuffle strain) that is capable of disulfide bond formation in the cytoplasm. Periplasmic expression of chFST315 using the pMAL-p5x vector system, which was designed to express maltose-binding protein (MBP) fusion protein, failed to produce a soluble recombinant protein. However, cytoplasmic expression of chFST315 using pMAL-c5x vector in SHuffle E. coli strain resulted in a soluble expression of the recombinant protein (MBP-chFST315). Combination of heparin and amylose resin affinity chromatography yielded about 6 mg/L purified MBP-chFST315. The purified MBP-chFST315 showed binding affinity to MSTN and activin in a pull-down assay, as well as inhibited MSTN and activin activity in an in vitro reporter gene assay. In conclusion, results of the study demonstrate that for the first time a recombinant, biologically active FST molecule can be produced in a soluble form in E. coli. The ability to produce FST in a cost-effective system is expected to allow us to investigate the potentials of FST as an agent to improve skeletal muscle growth of meat producing animals via suppression of MSTN.

  7. Characterization of three novel mechanosensitive channel activities in Escherichia coli.

    PubMed

    Edwards, Michelle D; Black, Susan; Rasmussen, Tim; Rasmussen, Akiko; Stokes, Neil R; Stephen, Terri-Leigh; Miller, Samantha; Booth, Ian R

    2012-01-01

    Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO and YjeP. The latter is the major component of the previously described MscM activity (~300 pS), while YnaI (~100 pS) and YbiO (~1000 pS) were previously unknown. Expression of native YbiO is NaCl-specific and RpoS-dependent. A Δ7 strain was created with all seven E. coli mechanosensitive channel genes deleted. High level expression of YnaI, YbiO or YjeP proteins from a multicopy plasmid in the Δ7 strain (MJFGH) leads to substantial protection against hypoosmotic shock. Purified homologs exhibit high molecular masses that are consistent with heptameric assemblies. This work reveals novel mechanosensitive channels and discusses the regulation of their expression in the context of possible additional functions.

  8. Azorean wild rabbits as reservoirs of antimicrobial resistant Escherichia coli.

    PubMed

    Marinho, Catarina; Igrejas, Gilberto; Gonçalves, Alexandre; Silva, Nuno; Santos, Tiago; Monteiro, Ricardo; Gonçalves, David; Rodrigues, Tiago; Poeta, Patrícia

    2014-12-01

    Antibiotic resistance in bacteria is an increasing problem that is not only constrained to the clinical setting but also to other environments that can lodge antibiotic resistant bacteria and therefore they may serve as reservoirs of genetic determinants of antibiotic resistance. One hundred and thirty-six faecal samples from European wild rabbits (Oryctolagus cuniculus algirus) were collected on São Jorge Island in Azores Archipelago, and analysed for Escherichia coli isolates. Seventy-seven isolates (56.6%) were recovered and studied for antimicrobial resistance, one isolate per positive sample. Thirteen (16.9%), 19 (24.7%), 25 (32.4%) and 20 (26%) isolates were ascribed to A, B1, B2 and D phylogenetic groups, respectively, by specific primer polymerase chain reaction. Different E. coli isolates were found to be resistant to ampicillin (16.9%), tetracycline (1.3%), streptomycin (42.9%), sulfamethoxazole-trimethoprim (1.3%), amikacin (1.3%), tobramycin (2.6%) and nalidixic acid (1.3%). Additionally, the blaTEM, tetA, strA/strB, aadA, sul1, intI, intI2 and qacEΔ+sul1 genes were found in most resistant isolates. This study showed that E. coli from the intestinal tract of wild rabbits from Azores Archipelago are resistant to widely prescribed antibiotics in medicine and they constitute a reservoir of antimicrobial resistant genes, which may play a significant role in the spread of antimicrobial resistance. Therefore, antibiotic resistant E. coli from Azorean wild rabbits may represent an ecological and public health problem.

  9. Characterization of fimbriae produced by enteropathogenic Escherichia coli.

    PubMed Central

    Girón, J A; Ho, A S; Schoolnik, G K

    1993-01-01

    Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC. Images PMID:7901197

  10. Inactivation kinetics of Escherichia coli by pulsed electron beam.

    PubMed

    Chalise, P R; Hotta, E; Matak, K E; Jaczynski, J

    2007-09-01

    A novel and compact low-energy (keV) high-power pulsed electron beam (e-beam) that utilizes a secondary emission electron gun (SEEG) was designed and constructed. Escherichia coli JM 109 at a concentration of 10(6) CFU/mL was spread-plated on Luria-Bertani (LB) medium and subjected to the SEEG e-beam. The e-beam was administered as 1 or 5 pulses. The duration of a single pulse was constant at 5 micros, e-beam current density was constant at 25 mA/cm2, and e-beam energy varied between 60 and 82.5 keV. Following treatment with the SEEG e-beam, survivors of the irradiated E. coli samples were enumerated by a standard 10-fold dilution and spread-plated. The survivor curves were plotted on logarithmic scale as a function of e-beam dose. The D10-values were calculated as a negative reciprocal of the slope of the survivor curves. The D10-values for E. coli inactivated with 1- and 5-pulse SEEG e-beam were 0.0026 and 0.0217 Gy, respectively. These D10-values were considerably lower than published D10-values for E. coli inactivated with conventional high-energy continuous e-beam, likely due to shorter exposure time (t), greater current density (J), and a pulse mode of the SEEG e-beam. The SEEG e-beam showed promising results for microbial inactivation in a nonthermal manner; however, due to low energy of the SEEG e-beam, current applications are limited to surface decontamination. The SEEG e-beam may be an efficient processing step for surface inactivation of food-borne pathogens on ready-to-eat products, including fresh and leafy vegetables.

  11. [Sensitivity to drugs of Escherichia coli strains isolated from poultry with coli septicemia].

    PubMed

    Giurov, B

    1985-01-01

    Investigations were carried out into the susceptibility of a total of 223 strains of Escherichia coli to therapeutic agents with the employment of the disk diffusion method. The organisms were isolated from internal organs and bone marrow of birds died of coli septicaemia. The serologic classification of the strains was defined with the use of 88 anti-group OK-agglutinating sera obtained through hyperimmunization of rabbits with the following Escherichia coli serotypes: 01-063, 068, 071, 073, 075, 078, 086, 0101, 0103, 0111-0114, 0119, 0124, 0129, 0135-0141, 0146, 0147, and 0149. It was found that serologically the strains referred as follows: 01-41 strains, 02-70 strains, 04-2 strains, 08-3 strains, 026-1 strain, 078-70 strains, 0111-2 strains, 0103-1 strain, 0141-1 strain. The number of untypable strains amounted to 32. Highest number of strains proved sensitive to colistin--96.06%, the remaining drugs following in a descending order: flumequine--95.65%, apramycin - 95.5%, gentamycin--93.72%, amoxicillin--93,8%, amikacin--88.57%, carbenicillin--86.88%, furazolidone--83,13%, and kanamycin--79.36%. High was the percent of strains resistant to tetracycline--66.17%, spectinomycin--61.67%, ampicillin--51.12%, chloramphenicol--50.23%, and streptomycin--44.84%.

  12. Uropathogenic Escherichia coli are less likely than paired fecal E. coli to have CRISPR loci.

    PubMed

    Dang, Trang Nguyen Doan; Zhang, Lixin; Zöllner, Sebastian; Srinivasan, Usha; Abbas, Khadija; Marrs, Carl F; Foxman, Betsy

    2013-10-01

    CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are short fragments of DNA that act as an adaptive immune system protecting bacteria against invasion by phages, plasmids or other forms of foreign DNA. Bacteria without a CRISPR locus may more readily adapt to environmental changes by acquiring foreign genetic material. Uropathogenic Escherichia coli (UPEC) live in a number of environments suggesting an ability to rapidly adapt to new environments. If UPEC are more adaptive than commensal E. coli we would expect that UPEC would have fewer CRISPR loci, and--if loci are present--that they would harbor fewer spacers than CRISPR loci in fecal E. coli. We tested this in vivo by comparing the number of CRISPR loci and spacers, and sensitivity to antibiotics (resistance is often obtained via plasmids) among 81 pairs of UPEC and fecal E. coli isolated from women with urinary tract infection. Each pair included one uropathogen and one commensal (fecal) sample from the same female patient. Fecal isolates had more repeats (p=0.009) and more unique spacers (p<0.0001) at four CRISPR loci than uropathogens. By contrast, uropathogens were more likely than fecal E. coli to be resistant to ampicillin, cefazolin and trimethoprim/sulfamethoxazole. However, no consistent association between CRISPRs and antibiotic resistance was identified. To our knowledge, this is the first study to compare fecal E. coli and pathogenic E. coli from the same individuals, and to test the association of CRISPR loci with antibiotic resistance. Our results suggest that the absence of CRISPR loci may make UPEC more susceptible to infection by phages or plasmids and allow them to adapt more quickly to various environments.

  13. Colonization with extraintestinal pathogenic Escherichia coli among nursing home residents and its relationship to fluoroquinolone resistance.

    PubMed

    Maslow, Joel N; Lautenbach, Ebbing; Glaze, Thomas; Bilker, Warren; Johnson, James R

    2004-09-01

    In a cross-sectional fecal prevalence survey involving 49 residents of a Veterans Affairs nursing home, 59% of subjects were colonized with extraintestinal pathogenic Escherichia coli (ExPEC), 22% were colonized with adhesin-positive E. coli, and 51% were colonized with fluoroquinolone-resistant E. coli. Among 80 unique isolates, adhesins correlated negatively and aerobactin correlated positively with fluoroquinolone resistance.

  14. A glimpse of Escherichia coli O157:H7 survival in soils from eastern China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7 (E. coli O157:H7) is an important food-borne pathogen, which continues to be a major public health concern worldwide. It is known that E. coli O157:H7 survive in soil environment might result in the contamination of fresh produce or water source. To investigate how the soils...

  15. Escherichia coli strain diversity: Selecting isolates for use as pathogen surrogates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Escherichia coli (E. coli) is commonly used as a surrogate for pathogens in research to identify sources of agricultural contamination and to characterize how pathogens persist on plant surfaces. However, E. coli strains are highly diverse, exhibiting differences in physical, chemical and...

  16. Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli (E. coli) food contaminations pose serious health and food safety concerns, and have been the subject of massive food recalls. Shiga toxin 2 (Stx2)-producing E. coli has been identified as the major cause of hemorrhagic colitis and hemolytic uremic syndrome (HUS), the most severe di...

  17. Detection of Escherichia Coli O157:H7 in Fecal Samples in Meat Goats

    ERIC Educational Resources Information Center

    Mobley, Ray; Madden, Uford; Brooks-Walter, Alexis

    2004-01-01

    Studies have reported the isolation of Escherichia coli (E. coli)O157:H7 from pork, lamb and poultry products, and from other animals including deer, horses, dogs, birds and humans. There is limited or no information on the presence of the organism in goats. The objectives of this study were to determine if E. coli O157:H7 was naturally occurring…

  18. Resistance of various shiga toxin-producing Escherichia coli to electrolyzed oxidizing water

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The resistance of thirty two strains of Escherichia coli O157:H7 and six major serotypes of non-O157 Shiga toxin- producing E. coli (STEC) plus E. coli O104 was tested against Electrolyzed oxidizing (EO) water using two different methods; modified AOAC 955.16 sequential inoculation method and minim...

  19. Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. Canine E. coli represents a good experimental model useful to study this pathology. Moreover, as des...

  20. A homolog of an Escherichia coli phosphate-binding protein gene from Xanthomonas oryzae pv. oryzae

    NASA Technical Reports Server (NTRS)

    Hopkins, C. M.; White, F. F.; Heaton, L. A.; Guikema, J. A.; Leach, J. E.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    A Xanthomonas oryzae pv. oryzae gene with sequence similarity to an Escherichia coli phosphate-binding protein gene (phoS) produces a periplasmic protein of apparent M(r) 35,000 when expressed in E. coli. Amino terminal sequencing revealed that a signal peptide is removed during transport to the periplasm in E. coli.

  1. Comparison of whole genome sequences from human and non-human Escherichia coli O26 strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) O26 is the second leading E. coli serogroup responsible for human illness outbreaks behind E. coli O157:H7. Recent outbreaks have been linked to emerging pathogenic O26:H11 strains harboring stx2 only. Cattle have been recognized as an important reserv...

  2. SURVIVAL OF ESCHERICHIA COLI 0157:H7 IN DAIRY CATTLE FEED WATER

    EPA Science Inventory

    Cattle feed waters from two dairy farms were used in a study to determine the survival characteristics of the bacterial pathogen Escherichia coli )157:H7 and wild-type E. coli. The E. coli 0157:H7 inoculum consisted of a consortium of isolates obtained from dairy cattle. Fresh ma...

  3. Proteomic differences between Escherichia coli strains that cause transient versus persistent intramammary infections [abstract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. Typically this infection is transient in nature and lasts 2-3 days. However, in a minority of cases, E. coli can cause a persistent intramammary infection. The mechanisms that enable certain strains of E. coli to cause a p...

  4. SILAC-based comparative analysis of pathogenic Escherichia coli secretomes.

    PubMed

    Boysen, Anders; Borch, Jonas; Krogh, Thøger Jensen; Hjernø, Karin; Møller-Jensen, Jakob

    2015-09-01

    Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative proteome analysis have the potential to discover both classes of proteins and hence form an important tool for discovering therapeutic targets. Adherent-invasive Escherichia coli (AIEC) and Enterotoxigenic E. coli (ETEC) are pathogenic variants of E. coli which cause intestinal disease in humans. AIEC is associated with Crohn's disease (CD), a chronic inflammatory condition of the gastrointestinal tract whereas ETEC is the major cause of human diarrhea which affects hundreds of millions annually. In spite of the disease burden associated with these pathogens, effective vaccines conferring long-term protection are still needed. In order to identify proteins with therapeutic potential, we have used mass spectrometry-based Stable Isotope Labeling with Amino acids in Cell culture (SILAC) quantitative proteomics method which allows us to compare the proteomes of pathogenic strains to commensal E. coli. In this study, we grew the pathogenic strains ETEC H10407, AIEC LF82 and the non-pathogenic reference strain E. coli K-12 MG1655 in parallel and used SILAC to compare protein levels in OMVs and culture supernatant. We have identified well-known virulence factors from both AIEC and ETEC, thus validating our experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the

  5. Engineering Escherichia coli to synthesize free fatty acids

    PubMed Central

    Lennen, Rebecca M.; Pfleger, Brian F.

    2013-01-01

    Fatty acid metabolism has received significant attention as a route for producing high-energy density, liquid transportation fuels and high-value oleochemicals from renewable feedstocks. If microbes can be engineered to produce these compounds at yields that approach the theoretical limits of 0.3–0.4 g/g glucose, then processes can be developed to replace current petrochemical technologies. Here, we review recent metabolic engineering efforts to maximize production of free fatty acids (FFA) in Escherichia coli, the first step towards production of downstream products. To date, metabolic engineers have succeeded in achieving higher yields of FFA than any downstream products. Regulation of fatty acid metabolism and the physiological effects of fatty acid production will also be reviewed from the perspective of identifying future engineering targets. PMID:23102412

  6. Phenotypic bistability in Escherichia coli's central carbon metabolism

    PubMed Central

    Kotte, Oliver; Volkmer, Benjamin; Radzikowski, Jakub L; Heinemann, Matthias

    2014-01-01

    Fluctuations in intracellular molecule abundance can lead to distinct, coexisting phenotypes in isogenic populations. Although metabolism continuously adapts to unpredictable environmental changes, and although bistability was found in certain substrate-uptake pathways, central carbon metabolism is thought to operate deterministically. Here, we combine experiment and theory to demonstrate that a clonal Escherichia coli population splits into two stochastically generated phenotypic subpopulations after glucose-gluconeogenic substrate shifts. Most cells refrain from growth, entering a dormant persister state that manifests as a lag phase in the population growth curve. The subpopulation-generating mechanism resides at the metabolic core, overarches the metabolic and transcriptional networks, and only allows the growth of cells initially achieving sufficiently high gluconeogenic flux. Thus, central metabolism does not ensure the gluconeogenic growth of individual cells, but uses a population-level adaptation resulting in responsive diversification upon nutrient changes. PMID:24987115

  7. Composition of cardiolipin molecular species in Escherichia coli.

    PubMed Central

    Yokota, K; Kanamoto, R; Kito, M

    1980-01-01

    The composition of the molecular species of acidic phospholipids in Escherichia coli B during the late exponential growth phase at 37 degrees C was determined. Two phosphatidyl groups of cardiolipin, the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties of cardiolipin, were isolated by limited hydrolysis with phospholipase C. No significant difference in the composition of the molecular species was found between the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties. On the other hand, the composition of the molecular species of phosphatidylglycerol was different from that of cardiolipin. Phosphatidylglycerol contained more of the 1-palmitoyl 2-cis-9,10-methylenehexadecanoyl and 1-palmitoyl 2-cis-11,12-methyleneoctadecanoyl species than did cardiolipin. The difference in the composition of the molecular species between cardiolipin and phosphatidylglycerol may depend on the difference in the turnover rates of both phospholipids. PMID:6988400

  8. Low Ubiquinone Content in Escherichia coli Causes Thiol Hypersensitivity

    PubMed Central

    Zeng, H.; Snavely, I.; Zamorano, P.; Javor, G. T.

    1998-01-01

    Thiol hypersensitivity in a mutant of Escherichia coli (IS16) was reversed by complementation with a plasmid that carried the ubiX gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal ubiX genes indicated that both parent and mutant strains were ubiX mutants. The low ubiquinone content of IS16 was possibly caused by a ubiD ubiX genotype. A ubiA mutant also exhibited thiol hypersensitivity. Neither IS16 nor the ubiA mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb− phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery. PMID:9658014

  9. Electron Microscopy of Chloramphenicol-treated Escherichia coli

    PubMed Central

    Morgan, Councilman; Rosenkranz, Herbert S.; Carr, Howard S.; Rose, Harry M.

    1967-01-01

    Thin sections of Escherichia coli were examined by electron microscopy at sequential intervals after addition and then removal of chloramphenicol. The first changes, occurring at 1 hr after exposure to the drug, were disappearance of the ribosomes and aggregation of the nuclear material toward the center of the bacteria. At 2 hr, aggregates of abnormal cytoplasmic granules first appeared and subsequently increased in size. By 23 hr, amorphous, electron-dense material had accumulated within, and at the periphery of, the nuclear matrix. With the removal of chloramphenicol, the bacteria became normal in appearance, passing through a series of stages that were sequential but not synchronous. At 145 min after removal of chloramphenicol, bacteria were encountered in the process of abnormal division. The influence of deoxyribonucleic acid and ribonucleic acid synthesis, and of energy metabolism, upon the changes seen electron microscopically in chloramphenicol-treated cells, was investigated by selectively inhibiting these functions with hydroxyurea, azauracil, and sodium azide, respectively. Images PMID:5337775

  10. Membrane protein production in Escherichia coli cell-free lysates.

    PubMed

    Henrich, Erik; Hein, Christopher; Dötsch, Volker; Bernhard, Frank

    2015-07-08

    Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.

  11. Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli*

    PubMed Central

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-01-01

    Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters. PMID:20525689

  12. Combinatorial method for overexpression of membrane proteins in Escherichia coli.

    PubMed

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-07-30

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

  13. Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli

    PubMed Central

    Lai, YuShuan; Rosenshine, Ilan; Leong, John M.; Frankel, Gad

    2013-01-01

    Enteropathogenic and enterohaemorrhagic Escherichia coli use a novel infection strategy to colonize the gut epithelium, involving translocation of their own receptor, Tir, via a type III secretion system and subsequent formation of attaching and effecting (A/E) lesions. Following integration into the host cell plasma membrane of cultured cells, and clustering by the outer membrane adhesin intimin, Tir triggers multiple actin polymerization pathways involving host and bacterial adaptor proteins that converge on the host Arp2/3 actin nucleator. Although initially thought to be involved in A/E lesion formation, recent data have shown that the known Tir-induced actin polymerization pathways are dispensable for this activity, but can play other major roles in colonization efficiency, in vivo fitness and systemic disease. In this review we summarize the roadmap leading from the discovery of Tir, through the different actin polymerization pathways it triggers, to our current understanding of their physiological functions. PMID:23927593

  14. Filling holes in peptidoglycan biogenesis of Escherichia coli.

    PubMed

    Ruiz, Natividad

    2016-12-01

    The peptidoglycan cell wall is an essential mesh-like structure in most bacteria. It is built outside the cytoplasmic membrane by polymerizing a disaccharide-pentapeptide into glycan chains that are crosslinked by peptides. The disaccharide-pentapeptide is synthetized as a lipid-linked precursor called lipid II, which is exported across the cytoplasmic membrane so that synthases can make new glycan chains. Growth of the peptidoglycan wall requires careful balancing of synthesis of glycan chains and hydrolysis of the preexisting structure to allow incorporation of new material. Recent studies in Escherichia coli have advanced our understanding of lipid II translocation across the membrane and how synthases are regulated to ensure proper envelope growth.

  15. Activity of murein hydrolases in synchronized cultures of Escherichia coli.

    PubMed Central

    Hakenbeck, R; Messer, W

    1977-01-01

    Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited. PMID:321419

  16. High-resolution structure of the Escherichia coli ribosome

    DOE PAGES

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; ...

    2015-03-16

    Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

  17. Programming a Pavlovian-like conditioning circuit in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Zhang, Haoqian; Lin, Min; Shi, Handuo; Ji, Weiyue; Huang, Longwen; Zhang, Xiaomeng; Shen, Shan; Gao, Rencheng; Wu, Shuke; Tian, Chengzhe; Yang, Zhenglin; Zhang, Guosheng; He, Siheng; Wang, Hao; Saw, Tiffany; Chen, Yiwei; Ouyang, Qi

    2014-01-01

    Synthetic genetic circuits are programmed in living cells to perform predetermined cellular functions. However, designing higher-order genetic circuits for sophisticated cellular activities remains a substantial challenge. Here we program a genetic circuit that executes Pavlovian-like conditioning, an archetypical sequential-logic function, in Escherichia coli. The circuit design is first specified by the subfunctions that are necessary for the single simultaneous conditioning, and is further genetically implemented using four function modules. During this process, quantitative analysis is applied to the optimization of the modules and fine-tuning of the interconnections. Analogous to classical Pavlovian conditioning, the resultant circuit enables the cells to respond to a certain stimulus only after a conditioning process. We show that, although the conditioning is digital in single cells, a dynamically progressive conditioning process emerges at the population level. This circuit, together with its rational design strategy, is a key step towards the implementation of more sophisticated cellular computing.

  18. De novo biosynthesis of Gastrodin in Escherichia coli.

    PubMed

    Bai, Yanfen; Yin, Hua; Bi, Huiping; Zhuang, Yibin; Liu, Tao; Ma, Yanhe

    2016-05-01

    Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545mgl(-1) gastrodin in 48h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.

  19. Collective motion in an active suspension of Escherichia coli bacteria

    NASA Astrophysics Data System (ADS)

    Gachelin, J.; Rousselet, A.; Lindner, A.; Clement, E.

    2014-02-01

    We investigate experimentally the emergence of collective motion in the bulk of an active suspension of Escherichia coli bacteria. When increasing the concentration from a dilute to a semi-dilute regime, we observe a continuous crossover from a dynamical cluster regime to a regime of ‘bio-turbulence’ convection patterns. We measure a length scale characterizing the collective motion as a function of the bacteria concentration. For bacteria fully supplied with oxygen, the increase of the correlation length is almost linear with concentration and at the largest concentrations tested, the correlation length could be as large as 24 bacterial body sizes (or 7-8 when including the flagella bundle). In contrast, under conditions of oxygen shortage the correlation length saturates at a value of around 7 body lengths.

  20. Curing of an R Factor from Escherichia coli by Trimethoprim

    PubMed Central

    Pinney, R. J.; Smith, J. T.

    1973-01-01

    R factor 1818, which we have shown previously to be eliminated by thymine starvation, was cured from three strains of Escherichia coli K-12 by overnight exposure to trimethoprim. Elimination was abolished in the presence of added thymine or thymidine, which suggests that curing is the result of the induction of thymineless conditions by trimethoprim. Starvation of the required amino acids proline and histidine had little effect on elimination, whereas methionine deprivation enhanced it. R factor curing was abolished by the presence of chloramphenicol, and it is concluded that protein synthesis is required for elimination to occur. It is suggested that elimination may result from the activity of a nuclease which is synthesized or induced during both direct thymine starvation and by trimethoprim treatment. PMID:4597737

  1. K99 surface antigen of Escherichia coli: antigenic characterization.

    PubMed Central

    Isaacson, R E

    1978-01-01

    K99 prepared by acid precipitation hemagglutinated guinea pig erythrocytes, whereas K99 prepared by chromatography on diethylaminoethyl-Sephadex did not. K99 purified by either procedure hemagglutinated horse erythrocytes. K99 prepared by acid precipitation contained a second antigen not presnet in the K99 prepared by chromatography on diethylaminoethyl-Sephadex. This antigen could be detected by immunoprecipitation with some, but not all, sera prepared against K99-positive Escherichia coli strains. It was assumed that this second antigen is not K99 and is responsible for the guinea pig erythrocyte hemagglutination reaction. Furthermore, the second antigen has an isoelectric point of 4.2, which has been reported by Morris and co-workers to be the isoelectric point of K99. Images PMID:83300

  2. Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy.

    PubMed

    Tang, Yulong; Li, Fengna; Tan, Bie; Liu, Gang; Kong, Xiangfeng; Hardwidge, Philip R; Yin, Yulong

    2014-06-25

    The morbidity and mortality in piglets caused by enterotoxigenic Escherichia coli (ETEC) results in large economic losses to the swine industry, but the precise pathogenesis of ETEC-associated diseases remains unknown. Intestinal epithelial cell autophagy serves as a host defense against pathogens. We found that ETEC induced autophagy, as measured by both the increased punctae distribution of GFP-LC3 and the enhanced conversion of LC3-I to LC3-II. Inhibiting autophagy resulted in decreased survival of IPEC-1 cells infected with ETEC. ETEC triggered autophagy in IPEC-1 cells through a pathway involving the mammalian target of rapamycin (mTOR), the extracellular signal-regulated kinases 1/2 (ERK1/2), and the AMP-activated protein kinase (AMPK).

  3. [Avian Escherichia coli virulence factors associated with coli septicemia in broiler chickens].

    PubMed

    Ramirez Santoyo, R M; Moreno Sala, A; Almanza Marquez, Y

    2001-01-01

    In order to detect phenotypic characteristics associated with pathogenicity, 25 strains of Escherichia coli, isolated from clinical cases of colisepticemia in broiler chickens, were examined to determine the following properties: colicinogenicity, colicin V production, type 1 fimbriae, hemolysin expression and motility. Colicinogenicity occurred in 72% of the strains, 56% of all strains produced colicin V, 84% were positive for type 1 fimbriae and 80% were positive for motility. None of the strains had hemolytic activity; however, all of them, expressed at least one of the other characteristics studied. These results suggest that the diversity of phenotypes detected partially explain the multifactorial nature of avian colisepticemia.

  4. A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias

    SciTech Connect

    Ruthenburg,A.; Graybosch, D.; Huetsch, J.; Verdine, G.

    2005-01-01

    DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped {beta}-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.

  5. Improvements In Ethanologenic Escherichia Coli and Klebsiella Oxytoca

    SciTech Connect

    Dr. David Nunn

    2010-09-30

    The current Verenium cellulosic ethanol process is based on the dilute-acid pretreatment of a biomass feedstock, followed by a two-stage fermentation of the pentose sugar-containing hydrolysate by a genetically modified ethanologenic Escherichia coli strain and a separate simultaneous saccharification-fermentation (SSF) of the cellulosic fraction by a genetically modified ethanologenic Klebsiella oxytoca strain and a fungal enzyme cocktail. In order to reduce unit operations and produce a fermentation beer with higher ethanol concentrations to reduce distillation costs, we have proposed to develop a simultaneous saccharification co-fermentation (SScF) process, where the fermentation of the pentose-containing hydrolysate and cellulosic fraction occurs within the same fermentation vessel. In order to accomplish this goal, improvements in the ethanologens must be made to address a number of issues that arise, including improved hydrolysate tolerance, co-fermentation of the pentose and hexose sugars and increased ethanol tolerance. Using a variety of approaches, including transcriptomics, strain adaptation, metagenomics and directed evolution, this work describes the efforts of a team of scientists from Verenium, University of Florida, Massachusetts Institute of Technology and Genomatica to improve the E. coli and K. oxytoca ethanologens to meet these requirements.

  6. A Murine Model for Escherichia coli Urinary Tract Infection

    PubMed Central

    Hannan, Thomas J.; Hunstad, David A.

    2015-01-01

    Urinary tract infections (UTI) are among the most common bacterial infections of humans. The mouse provides an excellent and tractable model system for cystitis and pyelonephritis caused by Escherichia coli and other uropathogens. Using a well-established model of experimental cystitis in which the bladders of female mice are infected via transurethral catheterization, the molecular details of the pathogenesis of bacterial cystitis have been substantially illuminated in the last decade. Uropathogenic E. coli attach to bladder epithelium (both in human and mouse) via adhesive type 1 pili, establish a replicative niche within epithelial cell cytoplasm, and form intracellular bacterial communities that are protected from antibiotic effects and immune clearance. The use of different inbred and mutant mouse strains offers the opportunity to study outcomes of infection, including resolution, formation of quiescent intracellular bacterial reservoirs, chronic bacterial cystitis, and recurrent infections. Urine, bladder, and kidney tissues can be analyzed by bacterial culture, histology, immunohistochemistry, immunofluorescent and confocal microscopy, electron microscopy, and flow cytometry, while a broad array of soluble markers (e.g., cytokines) can also be profiled in serum, urine, and tissue homogenates by ELISA, Western blotting, multiplex bead array, and other approaches. This model promises to afford continued opportunity for discovery of pathogenic mechanisms and evaluation of therapeutic and preventive strategies for acute, chronic, and recurrent UTI. PMID:26468108

  7. Association between antimicrobial consumption and resistance in Escherichia coli.

    PubMed

    Bergman, Miika; Nyberg, Solja T; Huovinen, Pentti; Paakkari, Pirkko; Hakanen, Antti J

    2009-03-01

    During a 9-year study period from 1997 through 2005, the association between antimicrobial resistance rates in Escherichia coli and outpatient antimicrobial consumption was investigated in 20 hospital districts in Finland. A total of 754,293 E. coli isolates, mainly from urine samples, were tested for antimicrobial resistance in 26 clinical microbiology laboratories. The following antimicrobials were studied: ampicillin, amoxicillin-clavulanate, cephalosporins, fluoroquinolones, trimethoprim, trimethoprim-sulfamethoxazole, pivmecillinam, and nitrofurantoin. We applied a protocol used in earlier studies in which the level of antimicrobial consumption over 1 year was compared with the level of resistance in the next year. Statistically significant associations were found for nitrofurantoin use versus nitrofurantoin resistance (P < 0.0001), cephalosporin use versus nitrofurantoin resistance (P = 0.0293), amoxicillin use versus fluoroquinolone resistance (P = 0.0031), and fluoroquinolone use versus ampicillin resistance (P = 0.0046). Interestingly, we found only a few associations between resistance and antimicrobial consumption. The majority of the associations studied were not significant, including the association between fluoroquinolone use and fluoroquinolone resistance.

  8. Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

    PubMed Central

    Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

    2014-01-01

    The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. PMID:24967819

  9. Recombinant expression of hydroxylated human collagen in Escherichia coli.

    PubMed

    Rutschmann, Christoph; Baumann, Stephan; Cabalzar, Jürg; Luther, Kelvin B; Hennet, Thierry

    2014-05-01

    Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.

  10. Properties of the Hemolytic Activities of Escherichia coli

    PubMed Central

    Short, Everett C.; Kurtz, Harold J.

    1971-01-01

    Some properties of the cell-free and cell-associated hemolysins of Escherichia coli were studied. Several strains of E. coli that were isolated from intestines of pigs with edema disease produce large quantities of cell-free hemolysin when grown in the presence of an extract of meat. The component of meat that stimulates production of cell-free hemolysin is not extracted by lipid solvents and is not dialyzable. The cell-free hemolysin is an acidic substance that occurs in two forms. It is inactivated by trypsin but not by lecithinase, lysozyme, ribonuclease, or deoxyribonuclease, shows optimum activity between pH 7 and 8, and requires calcium ion for activity. It does not appear to be an enzyme. The kinetics of the lytic reaction are most consistent with the hypothesis that one molecule of cell-free hemolysin is sufficient to lyse one erythrocyte and that it is inactivated in the lytic reaction. The cell-free hemolysin does not sufficiently damage the cell during the prelytic period to cause lysis after the hemolysin-calcium-erythrocyte complex has been disrupted. The cell-associated hemolysin was not separated from the cell by autolysis, freezing, sonic treatment, or treatment with trypsin or lysozyme. It appears to be closely associated with the metabolic status of the cell. Organisms that are highly hemolytic under usual conditions of assay immediately lose most of their hemolytic capability in the presence of sodium cyanide, streptomycin, nalidixic acid, and rifampin. PMID:16558036

  11. Evidence for antitermination in Escherichia coli RRNA transcription.

    PubMed

    Aksoy, S; Squires, C L; Squires, C

    1984-07-01

    The stable RNA operons of Escherichia coli do not exhibit polarity, even though they make an RNA product that is not translated. By contrast, most E. coli operons that specify proteins exhibit polarity if their translation is interrupted. The transcriptional component of this polarity depends on the action of Rho protein on the exposed mRNA, which results in premature transcription termination. Here we examine how a stable RNA operon (rrnG) transcript is protected from the Rho protein-mediated polarity response. We compared transcription from the ara and the rrnG promoters through a 16S DNA segment. In each case, the promoter-16S sequences were joined to a trp-lac fusion, and lacZ mRNA was examined in rho+ and rho-115 strains. We found significant Rho protein-dependent termination of transcripts from the ara promoter but little or no Rho protein effect on transcription from the rrnG promoter. We concluded that the transcript of the 16S ribosomal DNA segment does contain Rho protein-dependent transcription terminators, but there is an antitermination system in the rrnG control region that allows it to transcribe through those terminators.

  12. [Virulence factors and pathophysiology of extraintestinal pathogenic Escherichia coli].

    PubMed

    Bidet, P; Bonarcorsi, S; Bingen, E

    2012-11-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) causing urinary tract infections, bacteraemia or meningitis are characterized by a particular genetic background (phylogenetic group B2 and D) and the presence, within genetic pathogenicity islands (PAI) or plasmids, of genes encoding virulence factors involved in adhesion to epithelia, crossing of the body barriers (digestive, kidney, bloodbrain), iron uptake and resistance to the immune system. Among the many virulence factors described, two are particularly linked with a pathophysiological process: type P pili PapGII adhesin is linked with acute pyelonephritis, in the absence of abnormal flow of urine, and the K1 capsule is linked with neonatal meningitis. However, if the adhesin PapGII appears as the key factor of pyelonephritis, such that its absence in strain causing the infection is predictive of malformation or a vesico-ureteral reflux, the meningeal virulence of E. coli can not be reduced to a single virulence factor, but results from a combination of factors unique to each clone, and an imbalance between the immune defenses of the host and bacterial virulence.

  13. Interactions between chemotaxis genes and flagellar genes in Escherichia coli.

    PubMed Central

    Parkinson, J S; Parker, S R; Talbert, P B; Houts, S E

    1983-01-01

    Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias. Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci. Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus. When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias. Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations. This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action. However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products. These interactions may be instrumental in establishing the unstimulated swimming pattern of E. coli. Images PMID:6305913

  14. Proteomic adaptations to starvation prepare Escherichia coli for disinfection tolerance.

    PubMed

    Du, Zhe; Nandakumar, Renu; Nickerson, Kenneth W; Li, Xu

    2015-02-01

    Despite the low nutrient level and constant presence of secondary disinfectants, bacterial re-growth still occurs in drinking water distribution systems. The molecular mechanisms that starved bacteria use to survive low-level chlorine-based disinfectants are not well understood. The objective of this study is to investigate these molecular mechanisms at the protein level that prepare starved cells for disinfection tolerance. Two commonly used secondary disinfectants chlorine and monochloramine, both at 1 mg/L, were used in this study. The proteomes of normal and starved Escherichia coli (K12 MG1655) cells were studied using quantitative proteomics. Over 60-min disinfection, starved cells showed significantly higher disinfection tolerance than normal cells based on the inactivation curves for both chlorine and monochloramine. Proteomic analyses suggest that starvation may prepare cells for the oxidative stress that chlorine-based disinfection will cause by affecting glutathione metabolism. In addition, proteins involved in stress regulation and stress responses were among the ones up-regulated under both starvation and chlorine/monochloramine disinfection. By comparing the fold changes under different conditions, it is suggested that starvation prepares E. coli for disinfection tolerance by increasing the expression of enzymes that can help cells survive chlorine/monochloramine disinfection. Protein co-expression analyses show that proteins in glycolysis and pentose phosphate pathway that were up-regulated under starvation are also involved in disinfection tolerance. Finally, the production and detoxification of methylglyoxal may be involved in the chlorine-based disinfection and cell defense mechanisms.

  15. Proteomic Adaptations to Starvation Prepare Escherichia coli for Disinfection Tolerance

    PubMed Central

    Du, Zhe; Nandakumar, Renu; Nickerson, Kenneth; Li, Xu

    2015-01-01

    Despite the low nutrient level and constant presence of secondary disinfectants, bacterial re-growth still occurs in drinking water distribution systems. The molecular mechanisms that starved bacteria use to survive low-level chlorine-based disinfectants are not well understood. The objective of this study is to investigate these molecular mechanisms at the protein level that prepare starved cells for disinfection tolerance. Two commonly used secondary disinfectants chlorine and monochloramine, both at 1 mg/L, were used in this study. The proteomes of normal and starved Escherichia coli (K12 MG1655) cells were studied using quantitative proteomics. Over 60-min disinfection, starved cells showed significantly higher disinfection tolerance than normal cells based on the inactivation curves for both chlorine and monochloramine. Proteomic analyses suggest that starvation may prepare cells for the oxidative stress that chlorine-based disinfection will cause by affecting glutathione metabolism. In addition, proteins involved in stress regulation and stress responses were among the ones up-regulated under both starvation and chlorine/monochloramine disinfection. By comparing the fold changes under different conditions, it is suggested that starvation prepares E. coli for disinfection tolerance by increasing the expression of enzymes that can help cells survive chlorine/monochloramine disinfection. Protein co-expression analyses show that proteins in glycolysis and pentose phosphate pathway that were up-regulated under starvation are also involved in disinfection tolerance. Finally, the production and detoxification of methylglyoxal may be involved in the chlorine-based disinfection and cell defense mechanisms. PMID:25463932

  16. Behavioral responses of Escherichia coli to changes in redox potential.

    PubMed

    Bespalov, V A; Zhulin, I B; Taylor, B L

    1996-09-17

    Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential. In a spatial redox gradient of benzoquinone/benzoquinol, E. coli cells migrated to form a sharply defined band. Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band. This behavioral response was named redox taxis. Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force. The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector. The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis. A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis. A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein. A similar mechanism has been proposed for aerotaxis. Redox taxis may play an important role in the distribution of bacterial species in natural environments.

  17. Origins of chemoreceptor curvature sorting in Escherichia coli

    PubMed Central

    Draper, Will; Liphardt, Jan

    2017-01-01

    Bacterial chemoreceptors organize into large clusters at the cell poles. Despite a wealth of structural and biochemical information on the system's components, it is not clear how chemoreceptor clusters are reliably targeted to the cell pole. Here, we quantify the curvature-dependent localization of chemoreceptors in live cells by artificially deforming growing cells of Escherichia coli in curved agar microchambers, and find that chemoreceptor cluster localization is highly sensitive to membrane curvature. Through analysis of multiple mutants, we conclude that curvature sensitivity is intrinsic to chemoreceptor trimers-of-dimers, and results from conformational entropy within the trimer-of-dimers geometry. We use the principles of the conformational entropy model to engineer curvature sensitivity into a series of multi-component synthetic protein complexes. When expressed in E. coli, the synthetic complexes form large polar clusters, and a complex with inverted geometry avoids the cell poles. This demonstrates the successful rational design of both polar and anti-polar clustering, and provides a synthetic platform on which to build new systems. PMID:28322223

  18. Composite analysis for Escherichia coli at coastal beaches

    USGS Publications Warehouse

    Bertke, E.E.

    2007-01-01

    At some coastal beaches, concentrations of fecal-indicator bacteria can differ substantially between multiple points at the same beach at the same time. Because of this spatial variability, the recreational water quality at beaches is sometimes determined by stratifying a beach into several areas and collecting a sample from each area to analyze for the concentration of fecal-indicator bacteria. The average concentration of bacteria from those points is often used to compare to the recreational standard for advisory postings. Alternatively, if funds are limited, a single sample is collected to represent the beach. Compositing the samples collected from each section of the beach may yield equally accurate data as averaging concentrations from multiple points, at a reduced cost. In the study described herein, water samples were collected at multiple points from three Lake Erie beaches and analyzed for Escherichia coli on modified mTEC agar (EPA Method 1603). From the multiple-point samples, a composite sample (n = 116) was formed at each beach by combining equal aliquots of well-mixed water from each point. Results from this study indicate that E. coli concentrations from the arithmetic average of multiple-point samples and from composited samples are not significantly different (t = 1.59, p = 0.1139) and yield similar measures of recreational water quality; additionally, composite samples could result in a significant cost savings.

  19. Three mutations in Escherichia coli that generate transformable functional flagella.

    PubMed

    Wang, Wenjing; Jiang, Zhengzeng; Westermann, Martin; Ping, Liyan

    2012-11-01

    Hydrodynamics predicts that swimming bacteria generate a propulsion force when a helical flagellum rotates because rotating helices necessarily translate at a low Reynolds number. It is generally believed that the flagella of motile bacteria are semirigid helices with a fixed pitch determined by hydrodynamic principles. Here, we report the characterization of three mutations in laboratory strains of Escherichia coli that produce different steady-state flagella without losing cell motility. E. coli flagella rotate counterclockwise during forward swimming, and the normal form of the flagella is a left-handed helix. A single amino acid exchange A45G and a double mutation of A48S and S110A change the resting flagella to right-handed helices. The stationary flagella of the triple mutant were often straight or slightly curved at neutral pH. Deprotonation facilitates the helix formation of it. The helical and curved flagella can be transformed to the normal form by torsion upon rotation and thus propel the cell. These mutations arose in the long-term laboratory cultivation. However, flagella are under strong selection pressure as extracellular appendages, and similar transformable flagella would be common in natural environments.

  20. Endogenous ethanol affects biopolyester molecular weight in recombinant Escherichia coli.

    PubMed

    Hiroe, Ayaka; Hyakutake, Manami; Thomson, Nicholas M; Sivaniah, Easan; Tsuge, Takeharu

    2013-11-15

    In biopolyester synthesis, polyhydroxyalkanoate (PHA) synthase (PhaC) catalyzes the polymerization of PHA in bacterial cells, followed by a chain transfer (CT) reaction in which the PHA polymer chain is transferred from PhaC to a CT agent. Accordingly, the frequency of CT reaction determines PHA molecular weight. Previous studies have shown that exogenous alcohols are effective CT agents. This study aimed to clarify the effect of endogenous ethanol as a CT agent for poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis in recombinant Escherichia coli, by comparing with that of exogenous ethanol. Ethanol supplementation to the culture medium reduced P(3HB) molecular weights by up to 56% due to ethanol-induced CT reaction. NMR analysis of P(3HB) polymers purified from the culture supplemented with (13)C-labeled ethanol showed the formation of a covalent bond between ethanol and P(3HB) chain at the carboxyl end. Cultivation without ethanol supplementation resulted in the reduction of P(3HB) molecular weight with increasing host-produced ethanol depending on culture aeration. On the other hand, production in recombinant BW25113(ΔadhE), an alcohol dehydrogenase deletion strain, resulted in a 77% increase in molecular weight. Analysis of five E. coli strains revealed that the estimated number of CT reactions was correlated with ethanol production. These results demonstrate that host-produced ethanol acts as an equally effective CT agent as exogenous ethanol, and the control of ethanol production is important to regulate the PHA molecular weight.

  1. Light induced DEP for immobilizing and orienting Escherichia coli bacteria

    NASA Astrophysics Data System (ADS)

    Miccio, Lisa; Marchesano, Valentina; Mugnano, Martina; Grilli, Simonetta; Ferraro, Pietro

    2016-01-01

    Manipulating bacteria and understanding their behavior when interacting with different substrates are of fundamental importance for patterning, detection, and any other topics related to health-care, food-enterprise, etc. Here, we adopt an innovative dielectrophoretic (DEP) approach based on electrode-free DEP for investigating smart but simple strategies for immobilization and orientation of bacteria. Escherichia coli DH5-alpha strain has been selected as subject of the study. The light induced DEP is achieved through ferroelectric iron-doped lithium niobate crystals used as substrates. Due to the photorefractive (PR) property of such material, suitable light patterns allow writing spatial-charges-distribution inside its volume and the resultant electric fields are able to immobilize E. coli on the surface. The experiments showed that, after laser irradiation, about 80% of bacteria is blocked and oriented along a particular direction on the crystals within an area of few square centimeters. The investigation presented here could open the way for detection or patterning applications based on a new driving mechanism. Future perspectives also include the possibility to actively switch by light the DEP forces, through the writing/erasing characteristic of PR fields, to dynamically control biofilm spatial structure and arrangement.

  2. Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

    PubMed

    Loveridge, E Joel; Tey, Lai-Hock; Allemann, Rudolf K

    2010-01-27

    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties.

  3. Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme.

    PubMed

    Halford, S E; Schlesinger, M J; Gutfreund, H

    1972-03-01

    1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P(i) to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

  4. mcr-1 identified in Avian Pathogenic Escherichia coli (APEC)

    PubMed Central

    Lima Barbieri, Nicolle; Nielsen, Daniel W.; Wannemuehler, Yvonne; Cavender, Tia; Hussein, Ashraf; Yan, Shi-gan; Nolan, Lisa K.; Logue, Catherine M.

    2017-01-01

    Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide threatening the use of one of the most important antimicrobials for treating human disease. Here, we examined a collection (n = 980) of Avian Pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis from the US and internationally for the presence of mcr-1 and mcr-2, genes known to encode colistin resistance. Included in the analysis was an additional set of avian fecal E. coli (AFEC) (n = 220) isolates from healthy birds for comparative analysis. The mcr-1 gene was detected in a total of 12 isolates recovered from diseased production birds from China and Egypt. No mcr genes were detected in the healthy fecal isolates. The full mcr-1 gene from positive isolates was sequenced using specifically designed primers and were compared with sequences currently described in NCBI. mcr-1 positive isolates were also assessed for phenotypic colistin resistance and extended spectrum beta lactam phenotypes and genotypes. This study has identified mcr-1 in APEC isolates dating back to at least 2010 and suggests that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. PMID:28264015

  5. Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli.

    PubMed

    Vollmer, Waldemar; Bertsche, Ute

    2008-09-01

    The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.

  6. Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli

    PubMed Central

    Somavanshi, Rahul; Ghosh, Bhaswar; Sourjik, Victor

    2016-01-01

    The phosphotransferase system (PTS) plays a pivotal role in the uptake of multiple sugars in Escherichia coli and many other bacteria. In the cell, individual sugar-specific PTS branches are interconnected through a series of phosphotransfer reactions, thus creating a global network that not only phosphorylates incoming sugars but also regulates a number of cellular processes. Despite the apparent importance of the PTS network in bacterial physiology, the holistic function of the network in the cell remains unclear. Here we used Förster resonance energy transfer (FRET) to investigate the PTS network in E. coli, including the dynamics of protein interactions and the processing of different stimuli and their transmission to the chemotaxis pathway. Our results demonstrate that despite the seeming complexity of the cellular PTS network, its core part operates in a strikingly simple way, sensing the overall influx of PTS sugars irrespective of the sugar identity and distributing this information equally through all studied branches of the network. Moreover, it also integrates several other specific metabolic inputs. The integrated output of the PTS network is then transmitted linearly to the chemotaxis pathway, in stark contrast to the amplification of conventional chemotactic stimuli. Finally, we observe that default uptake through the uninduced PTS network correlates well with the quality of the carbon source, apparently representing an optimal regulatory strategy. PMID:27557415

  7. Spinach ferredoxin i: overproduction in Escherichia coli and purification.

    PubMed

    Piubelli, L; Aliverti, A; Bellintani, F; Zanetti, G

    1995-06-01

    Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts. A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction. Upon induction, ferredoxin I accounted for about 2.5% of soluble E. coli protein. A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed. The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine. Ferredoxin I was synthesized as the holoprotein, correctly assembled with the [2Fe-2S] cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase). The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy.

  8. Highly chlorinated Escherichia coli cannot be stained by propidium iodide.

    PubMed

    Phe, M-H; Dossot, M; Guilloteau, H; Block, J-C

    2007-05-01

    Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 micromol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.

  9. Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation.

    PubMed

    Majtan, Tomas; Frerman, Frank E; Kraus, Jan P

    2011-04-01

    Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe-S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.

  10. Calpain Mediates Epithelial Cell Microvillar Effacement by Enterohemorrhagic Escherichia Coli

    PubMed Central

    Lai, YuShuan; Riley, Kathleen; Cai, Andrew; Leong, John M.; Herman, Ira M.

    2011-01-01

    A member of the attaching and effacing (AE) family of pathogens, enterohemorrhagic Escherichia coli (EHEC) induces dramatic changes to the intestinal cell cytoskeleton, including effacement of microvilli. Effacement by the related pathogen enteropathogenic E. coli (EPEC) requires the activity of the Ca+2-dependent host protease, calpain, which participates in a variety of cellular processes, including cell adhesion and motility. We found that EHEC infection results in an increase in epithelial (CaCo-2a) cell calpain activity and that EHEC-induced microvillar effacement was blocked by ectopic expression of calpastatin, an endogenous calpain inhibitor, or by pretreatment of intestinal cells with a cell-penetrating version of calpastatin. In addition, ezrin, a known calpain substrate that links the plasma membrane to axial actin filaments in microvilli, was cleaved in a calpain-dependent manner during EHEC infection and lost from its normal locale within microvilli. Calpain may be a central conduit through which EHEC and other AE pathogens induce enterocyte cytoskeletal remodeling and exert their pathogenic effects. PMID:22073041

  11. Dynamic organization of chromosomal DNA in Escherichia coli.

    PubMed

    Niki, H; Yamaichi, Y; Hiraga, S

    2000-01-15

    We have revealed the subcellular localization of different DNA segments that are located at approximately 230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH). The series of chromosome segments is localized within the cell in the same order as the chromosome map. The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain. In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region. The segment also shows similar localization patterns, which we call the Ter domain. In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles. Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell. An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells. These studies thus suggest that the E. coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome.

  12. Enhanced succinate production from glycerol by engineered Escherichia coli strains.

    PubMed

    Li, Qing; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-10-01

    In this study, an engineered strain Escherichia coli MLB (ldhA(-)pflB(-)) was constructed for production of succinate from glycerol. The succinate yield was 0.37mol/mol in anaerobic culture, however, the growth and glycerol consumption rates were very slow, resulting in a low succinate level. Two-stage fermentation was performed in flasks, and the succinate yield reached 0.93mol/mol, but the succinate titer was still low. Hence, overexpression of malate dehydrogenase, malic enzyme, phosphoenolpyruvate (PEP) carboxylase and PEP carboxykinase (PCK) from E. coli, and pyruvate carboxylase from Corynebacterium glutamicum in MLB was investigated for improving succinate production. Overexpression of PCK resulted in remarkable enhancement of glycerol consumption and succinate production. In flask experiments, the succinate concentration reached 118.1mM, and in a 1.5-L bioreactor the succinate concentration further increased to 360.2mM. The highest succinate yield achieved 0.93mol/mol, which was 93% of the theoretical yield, in the anaerobic stage.

  13. Simple method for purification of enterotoxigenic Escherichia coli fimbriae.

    PubMed

    Curtis, Brittany; Grassel, Christen; Laufer, Rachel S; Sears, Khandra T; Pasetti, Marcela F; Barry, Eileen M; Simon, Raphael

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera.

  14. Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

    PubMed Central

    Hebbelstrup Jensen, Betina; Olsen, Katharina E. P.; Struve, Carsten; Petersen, Andreas Munk

    2014-01-01

    SUMMARY Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity. PMID:24982324

  15. Proton translocation in cytochrome-deficient mutants of Escherichia coli.

    PubMed Central

    Brookman, J J; Downie, J A; Gibson, F; Cox, G B; Rosenberg, H

    1979-01-01

    Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase. PMID:154508

  16. Induction and control of the autolytic system of Escherichia coli.

    PubMed Central

    Leduc, M; Kasra, R; van Heijenoort, J

    1982-01-01

    Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time. For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments. The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine. The different autolyses examined shared certain common properties. In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process. However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells. Autolysis of growing cells required RNA, protein, and fatty acid synthesis. No such requirements were observed with shock-induced autolysis performed with harvested cells. Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E. Coli. Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis. PMID:6181050

  17. Public Health Microbiology of Shiga Toxin-Producing Escherichia coli.

    PubMed

    Caprioli, Alfredo; Scavia, Gaia; Morabito, Stefano

    2014-12-01

    Shiga toxin-producing Escherichia coli (STEC) strains are the only pathogenic group of E. coli that has a definite zoonotic origin, with ruminants and, in particular, cattle being recognized as the major reservoir. Most human STEC infections are food borne, but the routes of transmission include direct contact with animals and a variety of environment-related exposures. Therefore, STEC public health microbiology spans the fields of medical, veterinary, food, water, and environmental microbiology, requiring a "One Health" perspective and laboratory scientists with the ability to work effectively across disciplines. Public health microbiology laboratories play a central role in the surveillance of STEC infections, as well as in the preparedness for responding to outbreaks and in providing scientific evidence for the implementation of prevention and control measures. This article reviews (i) how the integration of surveillance of STEC infections and monitoring of these pathogens in animal reservoirs and potential food vehicles may contribute to their control; (ii) the role of reference laboratories, in both the public health and veterinary and food sectors; and (iii) the public health perspectives, including those related to regulatory issues in both the European Union and the United States.

  18. Structure and Biochemical Activities of Escherichia coli MgsA

    SciTech Connect

    Page, Asher N.; George, Nicholas P.; Marceau, Aimee H.; Cox, Michael M.; Keck, James L.

    2012-02-27

    Bacterial 'maintenance of genome stability protein A' (MgsA) and related eukaryotic enzymes play important roles in cellular responses to stalled DNA replication processes. Sequence information identifies MgsA enzymes as members of the clamp loader clade of AAA{sup +} proteins, but structural information defining the family has been limited. Here, the x-ray crystal structure of Escherichia coli MgsA is described, revealing a homotetrameric arrangement for the protein that distinguishes it from other clamp loader clade AAA{sup +} proteins. Each MgsA protomer is composed of three elements as follows: ATP-binding and helical lid domains (conserved among AAA{sup +} proteins) and a tetramerization domain. Although the tetramerization domains bury the greatest amount of surface area in the MgsA oligomer, each of the domains participates in oligomerization to form a highly intertwined quaternary structure. Phosphate is bound at each AAA{sup +} ATP-binding site, but the active sites do not appear to be in a catalytically competent conformation due to displacement of Arg finger residues. E. coli MgsA is also shown to form a complex with the single-stranded DNA-binding protein through co-purification and biochemical studies. MgsA DNA-dependent ATPase activity is inhibited by single-stranded DNA-binding protein. Together, these structural and biochemical observations provide insights into the mechanisms of MgsA family AAA{sup +} proteins.

  19. Enhanced hydrogen production from glucose by metabolically engineered Escherichia coli.

    PubMed

    Maeda, Toshinari; Sanchez-Torres, Viviana; Wood, Thomas K

    2007-12-01

    To utilize fermentative bacteria for producing the alternative fuel hydrogen, we performed successive rounds of P1 transduction from the Keio Escherichia coli K-12 library to introduce multiple, stable mutations into a single bacterium to direct the metabolic flux toward hydrogen production. E. coli cells convert glucose to various organic acids (such as succinate, pyruvate, lactate, formate, and acetate) to synthesize energy and hydrogen from formate by the formate hydrogen-lyase (FHL) system that consists of hydrogenase 3 and formate dehydrogenase-H. We altered the regulation of FHL by inactivating the repressor encoded by hycA and by overexpressing the activator encoded by fhlA, removed hydrogen uptake activity by deleting hyaB (hydrogenase 1) and hybC (hydrogenase 2), redirected glucose metabolism to formate by using the fdnG, fdoG, narG, focA, focB, poxB, and aceE mutations, and inactivated the succinate and lactate synthesis pathways by deleting frdC and ldhA, respectively. The best of the metabolically engineered strains, BW25113 hyaB hybC hycA fdoG frdC ldhA aceE, increased hydrogen production 4.6-fold from glucose and increased the hydrogen yield twofold from 0.65 to 1.3 mol H(2)/mol glucose (maximum, 2 mol H(2)/mol glucose).

  20. Molecular Evolution of the Escherichia Coli Chromosome. IV. Sequence Comparisons

    PubMed Central

    Milkman, R.; Bridges, M. M.

    1993-01-01

    DNA sequences have been compared in a 4,400-bp region for Escherichia coli K12 and 36 ECOR strains. Discontinuities in degree of similarity, previously inferred, are confirmed in detail. Three clonal frames are described on the basis of the present local high-resolution data, as well as previous analyses of restriction fragment length polymorphism (RFLP) and of multilocus enzyme electrophoresis (MLEE) covering small regions more widely dispersed on the chromosome. These three approaches show important consistency. The data illustrate the fact that, in the limited context of intraspecific genomic sequence variation, clonality and homology are synonymous. Two estimable quantitative properties are defined: recency of common ancestry (the reciprocal of the log(10) of the number of generations since the most recent common ancestor), and the number of nucleotide pairs over which a given recency of common ancestry applies. In principle, these parameters are measures of the degree and physical extent of homology. The small size of apparent recombinational replacements, together with the observation that they occasionally occur in discontinuous series, raises the question of whether they result from the superimposition of replacements of much larger size (as expected from an elementary interpretation of conjugation and transduction in experimental E. coli systems) or via an alternative mechanism. Length polymorphisms of several sorts are described. PMID:8095913