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Sample records for escherichia coli ribosomal

  1. High-resolution structure of the Escherichia coli ribosome

    PubMed Central

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; Altman, Roger B.; Blanchard, Scott C.; Cate, Jamie H. D.

    2015-01-01

    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. 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. PMID:25775265

  2. Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes.

    PubMed

    Soung, George Y; Miller, Jennifer L; Koc, Hasan; Koc, Emine C

    2009-07-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in 24 Escherichia coli ribosomal proteins by tandem mass spectrometry. Detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining, and antibodies for phospho-Ser, Thr, and Tyr; or by mass spectrometry equipped with a capability to detect addition and loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, and L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given phosphorylation sites in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  3. Time-resolved binding of azithromycin to Escherichia coli ribosomes.

    PubMed

    Petropoulos, Alexandros D; Kouvela, Ekaterini C; Starosta, Agata L; Wilson, Daniel N; Dinos, George P; Kalpaxis, Dimitrios L

    2009-01-30

    Azithromycin is a semisynthetic derivative of erythromycin that inhibits bacterial protein synthesis by binding within the peptide exit tunnel of the 50S ribosomal subunit. Nevertheless, there is still debate over what localization is primarily responsible for azithromycin binding and as to how many molecules of the drug actually bind per ribosome. In the present study, kinetic methods and footprinting analysis are coupled together to provide time-resolved details of the azithromycin binding process. It is shown that azithromycin binds to Escherichia coli ribosomes in a two-step process: The first-step involves recognition of azithromycin by the ribosomal machinery and places the drug in a low-affinity site located in the upper part of the exit tunnel. The second step corresponds to the slow formation of a final complex that is both much tighter and more potent in hindering the progression of the nascent peptide through the exit tunnel. Substitution of uracil by cytosine at nucleoside 2609 of 23S rRNA, a base implicated in the high-affinity site, facilitates the shift of azithromycin to this site. In contrast, mutation U754A hardly affects the binding process. Binding of azithromycin to both sites is hindered by high concentrations of Mg(2+) ions. Unlike Mg(2+) ions, polyamines do not significantly affect drug binding to the low-affinity site but attenuate the formation of the final complex. The low- and high-affinity sites of azithromycin binding are mutually exclusive, which means that one molecule of the drug binds per E. coli ribosome at a time. In contrast, kinetic and binding data indicate that in Deinococcus radiodurans, two molecules of azithromycin bind cooperatively to the ribosome. This finding confirms previous crystallographic results and supports the notion that species-specific structural differences may primarily account for the apparent discrepancies between the antibiotic binding modes obtained for different organisms. PMID:19071138

  4. Ribosome Patterns in Escherichia coli Growing at Various Rates

    PubMed Central

    Varricchio, Frederick; Monier, Roger

    1971-01-01

    The distribution of ribosomes, 30 and 50S subunits and polysomes, at three different growth rates of Escherichia coli strains B and K-12 has been studied. The usual percentage of subunits is about 20%. However, at the lowest growth rate (μ = generations/hour), μ = 0.45 at 30C, the proportion of subunits is about 30%. An exceptional situation exists in K-12 strains growing at maximum growth rate, μ = 1.35, where the percentage of subunits is 45%. Several points of control over ribosome production are thus indicated. It is suggested that “subunit pool” is essentially a reserve. Furthermore, the polysome content when related to deoxyribonucleic acid content varies directly with the growth rate, which indicates the average efficiency of polysomes in protein synthesis does not vary over the range of growth rates tested. PMID:5001192

  5. Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.

    PubMed Central

    Girbés, T; Barbieri, L; Ferreras, M; Arias, F J; Rojo, M A; Iglesias, R; Alegre, C; Escarmis, C; Stirpe, F

    1993-01-01

    The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations. Images PMID:8407849

  6. All-atom homology model of the Escherichia coli 30S ribosomal subunit.

    PubMed

    Tung, Chang-Shung; Joseph, Simpson; Sanbonmatsu, Kevin Y

    2002-10-01

    Understanding the structural basis of ribosomal function requires close comparison between biochemical and structural data. Although a large amount of biochemical data are available for the Escherichia coli ribosome, the structure has not been solved to atomic resolution. Using a new RNA homology procedure, we have modeled the all-atom structure of the E. coli 30S ribosomal subunit. We find that the tertiary structure of the ribosome core, including the A-, P- and E-sites, is highly conserved. The hypervariable regions in our structure, which differ from the structure of the 30S ribosomal subunit from Thermus thermophilus, are consistent with the cryo-EM map of the E. coli ribosome. PMID:12244297

  7. The fail-safe system to rescue the stalled ribosomes in Escherichia coli

    PubMed Central

    Abo, Tatsuhiko; Chadani, Yuhei

    2014-01-01

    Translation terminates at stop codon. Without stop codon, ribosome cannot terminate translation properly and reaches and stalls at the 3′-end of the mRNA lacking stop codon. Bacterial tmRNA-mediated trans-translation releases such stalled ribosome and targets the protein product to degradation by adding specific “degradation tag.” Recently two alternative ribosome rescue factors, ArfA (YhdL) and ArfB (YaeJ), have been found in Escherichia coli. These three ribosome rescue systems are different each other in terms of molecular mechanism of ribosome rescue and their activity, but they are mutually related and co-operate to maintain the translation system in shape. This suggests the biological significance of ribosome rescue. PMID:24782844

  8. Most RNAs regulating ribosomal protein biosynthesis in Escherichia coli are narrowly distributed to Gammaproteobacteria

    PubMed Central

    Fu, Yang; Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M.

    2013-01-01

    In Escherichia coli, 12 distinct RNA structures within the transcripts encoding ribosomal proteins interact with specific ribosomal proteins to allow autogenous regulation of expression from large multi-gene operons, thus coordinating ribosomal protein biosynthesis across multiple operons. However, these RNA structures are typically not represented in the RNA Families Database or annotated in genomic sequences databases, and their phylogenetic distribution is largely unknown. To investigate the extent to which these RNA structures are conserved across eubacterial phyla, we created multiple sequence alignments representing 10 of these messenger RNA (mRNA) structures in E. coli. We find that while three RNA structures are widely distributed across many phyla of bacteria, seven of the RNAs are narrowly distributed to a few orders of Gammaproteobacteria. To experimentally validate our computational predictions, we biochemically confirmed dual L1-binding sites identified in many Firmicute species. This work reveals that RNA-based regulation of ribosomal protein biosynthesis is used in nearly all eubacterial phyla, but the specific RNA structures that regulate ribosomal protein biosynthesis in E. coli are narrowly distributed. These results highlight the limits of our knowledge regarding ribosomal protein biosynthesis regulation outside of E. coli, and the potential for alternative RNA structures responsible for regulating ribosomal proteins in other eubacteria. PMID:23396277

  9. Kinetics of paused ribosome recycling in Escherichia coli

    PubMed Central

    Janssen, Brian D.; Hayes, Christopher S.

    2009-01-01

    Summary The bacterial tmRNA•SmpB system recycles stalled translation complexes in a process termed ‘ribosome rescue’. tmRNA•SmpB specifically recognizes ribosomes that are paused at or near the 3′ end of truncated mRNA, and therefore nucleolytic mRNA processing is required before paused ribosomes can be rescued from full-length transcripts. Here, we examine the recycling of ribosomes paused on both full-length and truncated mRNAs. Peptidyl-tRNAs corresponding to each paused translation complex were identified, and their turnover kinetics used to estimate the half-lives of paused ribosomes in vivo. Ribosomes were paused at stop codons on full-length mRNA using a nascent peptide motif that interferes with translation termination and elicits tmRNA•SmpB activity. Peptidyl-tRNA turnover from these termination-paused ribosomes was slightly more rapid in tmRNA+ cells (T1/2 = 22 ± 2.2 s), compared to ΔtmRNA cells (T1/2 = 32 ± 1.6 s). Overexpression of release factor-1 (RF-1) greatly accelerated peptidyl-tRNA turnover from termination-paused ribosomes in both tmRNA+ and ΔtmRNA cells, whereas other termination factors had little or no effect on recycling. In contrast to inefficient translation termination, ribosome recycling from truncated transcripts lacking in-frame stop codons was dramatically accelerated by tmRNA•SmpB. However, peptidyl-tRNA still turned over from nonstop-paused ribosomes at a significant rate (t1/2 = 61 ± 7.3 s) in ΔtmRNA cells. Overexpression of RF-1, RF-3, and ribosome recycling factor (RRF) in ΔtmRNA cells failed to accelerate ribosome recycling from nonstop mRNA. These results indicate that tmRNA•SmpB activity is rate-limited by mRNA cleavage, and that RF-3 and RRF do not constitute a tmRNA-independent rescue pathway as previously suggested. Peptidyl-tRNA turnover from nonstop-paused ribosomes in ΔtmRNA cells suggests the existence of another uncharacterized ribosome rescue pathway. PMID:19761774

  10. Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

    PubMed Central

    Kitahara, Kei; Yasutake, Yoshiaki; Miyazaki, Kentaro

    2012-01-01

    The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome’s structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, we have recently shown that an active hybrid ribosome whose 16S rRNA has been specifically substituted with that from non–E. coli bacteria can be reconstituted in vivo. To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality, we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E. coli Δ7. Various functional genes were obtained from the Gammaproteobacteria and Betaproteobacteria lineages. Despite the large sequence diversity (80.9–99.0% identity with E. coli 16S rRNA) of the functional 16S rRNA molecules, the doubling times (DTs) of each mutant increased only modestly with decreasing sequence identity (average increase in DT, 4.6 s per mutation). The three-dimensional structure of the 30S ribosome showed that at least 40.7% (628/1,542) of the nucleotides were variable, even at ribosomal protein-binding sites, provided that the secondary structures were properly conserved. Our results clearly demonstrate that 16S rRNA functionality largely depends on the secondary structure but not on the sequence itself. PMID:23112186

  11. Breakdown by Streptomycin of Initiation Complexes Formed on Ribosomes of Escherichia coli*

    PubMed Central

    Modolell, Juan; Davis, Bernard D.

    1970-01-01

    Streptomycin induces breakdown of the completed 70S initiation complex on ribosomes of Escherichia coli, but it does not interfere with any step in the formation of the complex. Moreover, it does not appear to interact with the ribosome in any special way during initiation, since the kinetics of breakdown are the same whether streptomycin is added before formation of the initiation complex, or after its completion, or (as previously observed) after formation of a polypeptide. fMet-tRNA is released as such, without chain elongation; it is released from a puromycin-reactive („P”) site. Streptomycin thus appears to distort not only the A site of the ribosome (as suggested earlier) but also the P site. PMID:4922285

  12. Markerless Escherichia coli rrn Deletion Strains for Genetic Determination of Ribosomal Binding Sites.

    PubMed

    Quan, Selwyn; Skovgaard, Ole; McLaughlin, Robert E; Buurman, Ed T; Squires, Catherine L

    2015-12-01

    Single-copy rrn strains facilitate genetic ribosomal studies in Escherichia coli. Consecutive markerless deletion of rrn operons resulted in slower growth upon inactivation of the fourth copy, which was reversed by supplying transfer RNA genes encoded in rrn operons in trans. Removal of the sixth, penultimate rrn copy led to a reduced growth rate due to limited rrn gene dosage. Whole-genome sequencing of variants of single-copy rrn strains revealed duplications of large stretches of genomic DNA. The combination of selective pressure, resulting from the decreased growth rate, and the six identical remaining scar sequences, facilitating homologous recombination events, presumably leads to elevated genomic instability. PMID:26438293

  13. Binding of Dihydrostreptomycin to Escherichia coli Ribosomes: Characteristics and Equilibrium of the Reaction

    PubMed Central

    Chang, F. N.; Flaks, Joel G.

    1972-01-01

    The binding of dihydrostreptomycin to ribosomes and ribosomal subunits of a number of different Escherichia coli strains was studied, and the Mg2+ and pH dependence, as well as the effect of salts and polynucleotides, was determined. The only requirement for binding with ribosomes and subunits from susceptible strains was 10 mm Mg2+. Monovalent salts weakened the binding in a manner similar to the effects on ribonucleic acid secondary structure, and this was antagonized to some extent by increased amounts of Mg2+. Bound dihydrostreptomycin could be readily exchanged by streptomycin and any antibiotically active derivative, but not by fragments of the antibiotic or any other aminoglycoside. With native (run-off) 70S ribosomes from streptomycin-susceptible strains, the binding was rapid and relatively temperature independent over the range from 0 to 37 C. Polynucleotides did not stimulate the binding. With concentrations of dihydrostreptomycin up to 10−5m, greater than 95% of native 70S ribosomes bound exactly 1 molecule of the antibiotic tightly, with a Kdiss for the bound complex at 25 C of 9.4 × 10−8m. The following thermodynamic parameters were found for the binding with 70S ribosomes at 25 C:ΔG° = −9.6 kcal/mole, ΔH° = −6.2 kcal/mole, and ΔS° = +11.4 entropy units/mole. Differences in affinity for the antibiotic were found between ribosomes of K-12 strains and those of other E. coli strains. There was insignificant binding to 70S ribosomes or subunits from streptomycin-resistant or -dependent strains, and to 50S subunits from susceptible strains. The binding to 30S subunits from susceptible strains was weaker by an order of magnitude than that to the 70S particle, with a Kdiss at 25 C of 10−6m. Polyuridylic acid stimulated this binding slightly but did not influence the affinity of the bound molecule. At antibiotic concentrations above 10−5m, streptomycin-susceptible 70S and 30S particles bound additional molecules of the antibiotic, and

  14. Insights into the mechanism of azithromycin interaction with an Escherichia coli functional ribosomal complex.

    PubMed

    Dinos, G P; Michelinaki, M; Kalpaxis, D L

    2001-06-01

    Azithromycin, a derivative of erythromycin with improved activity against Gram-negative bacteria, exhibits a marginal inhibition effect in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. This renders the study of azithromycin interaction with Ac[(3)H]Phe-tRNA. poly(U). 70S ribosome complex (complex C) impossible, if we analyze its effect on peptide bond formation. To overcome this problem, we have used an alternative approach to investigate kinetically the azithromycin interaction with complex C and to compare the azithromycin binding properties with those of erythromycin. This approach was based on the ability of azithromycin to compete with tylosin, a macrolide antibiotic strongly inhibiting the puromycin reaction. Detailed kinetic analysis revealed that the encounter complex CA between complex C and azithromycin (A) undergoes a slow isomerization to a tighter complex C*A, which remains active toward puromycin. The determination of inhibition and isomerization rate constants enabled us to classify azithromycin as a slow-binding ligand of ribosomes. Compared with erythromycin, azithromycin is a better inducer and stabilizer of the C*A complex. This finding may explain the superiority of azithromycin as inhibitor of translation in E. coli cells and many other Gram-negative bacteria. PMID:11353804

  15. In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

    PubMed Central

    Yates, J L; Arfsten, A E; Nomura, M

    1980-01-01

    Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins. Images PMID:6445562

  16. Stepwise binding of tylosin and erythromycin to Escherichia coli ribosomes, characterized by kinetic and footprinting analysis.

    PubMed

    Petropoulos, Alexandros D; Kouvela, Ekaterini C; Dinos, George P; Kalpaxis, Dimitrios L

    2008-02-22

    Erythromycin and tylosin are 14- and 16-membered lactone ring macrolides, respectively. The current work shows by means of kinetic and chemical footprinting analysis that both antibiotics bind to Escherichia coli ribosomes in a two-step process. The first step established rapidly, involves a low-affinity binding site placed at the entrance of the exit tunnel in the large ribosomal subunit, where macrolides bind primarily through their hydrophobic portions. Subsequently, slow conformational changes mediated by the antibiotic hydrophilic portion push the drugs deeper into the tunnel, in a high-affinity site. Compared with erythromycin, tylosin shifts to the high-affinity site more rapidly, due to the interaction of the mycinose sugar of the drug with the loop of H35 in domain II of 23 S rRNA. Consistently, mutations of nucleosides U2609 and U754 implicated in the high-affinity site reduce the shift of tylosin to this site and destabilize, respectively, the final drug-ribosome complex. The weak interaction between tylosin and the ribosome is Mg2+ independent, unlike the tight binding. In contrast, both interactions between erythromycin and the ribosome are reduced by increasing concentrations of Mg2+ ions. Polyamines attenuate erythromycin affinity for the ribosome at both sequential steps of binding. In contrast, polyamines facilitate the initial binding of tylosin, but exert a detrimental, more pronounced, effect on the drug accommodation at its final position. Our results emphasize the role of the particular interactions that side chains of tylosin and erythromycin establish with 23 S rRNA, which govern the exact binding process of each drug and its response to the ionic environment. PMID:18079110

  17. Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

    PubMed Central

    Yamagishi, M; Nomura, M

    1988-01-01

    Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly. PMID:3053641

  18. Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

    PubMed

    Vanet, A; Plumbridge, J A; Guérin, M F; Alix, J H

    1994-12-01

    The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable. PMID:7715456

  19. pH-dependent structural changes of helix 69 from Escherichia coli 23S ribosomal RNA

    PubMed Central

    Abeysirigunawardena, Sanjaya C.; Chow, Christine S.

    2008-01-01

    Helix 69 in 23S rRNA is a region in the ribosome that participates in a considerable number of RNA–RNA and RNA–protein interactions. Conformational flexibility is essential for such a region to interact and accommodate protein factors at different stages of protein biosynthesis. In this study, pH-dependent structural and stability changes were observed for helix 69 through a variety of spectroscopic techniques, such as circular dichroism spectroscopy, UV melting, and nuclear magnetic resonance spectroscopy. In Escherichia coli 23S rRNA, helix 69 contains pseudouridine residues at positions 1911, 1915, and 1917. The presence of these pseudouridines was found to be essential for the pH-induced conformational changes. Some of the pH-dependent changes appear to be localized to the loop region of helix 69, emphasizing the importance of the highly conserved nature of residues in this region. PMID:18268024

  20. Analysis of tetra- and hepta-nucleotides motifs promoting -1 ribosomal frameshifting in Escherichia coli

    PubMed Central

    Sharma, Virag; Prère, Marie-Françoise; Canal, Isabelle; Firth, Andrew E.; Atkins, John F.; Baranov, Pavel V.; Fayet, Olivier

    2014-01-01

    Programmed ribosomal -1 frameshifting is a non-standard decoding process occurring when ribosomes encounter a signal embedded in the mRNA of certain eukaryotic and prokaryotic genes. This signal has a mandatory component, the frameshift motif: it is either a Z_ZZN tetramer or a X_XXZ_ZZN heptamer (where ZZZ and XXX are three identical nucleotides) allowing cognate or near-cognate repairing to the -1 frame of the A site or A and P sites tRNAs. Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%. The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ. The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting. PMID:24875478

  1. Analysis of tetra- and hepta-nucleotides motifs promoting -1 ribosomal frameshifting in Escherichia coli.

    PubMed

    Sharma, Virag; Prère, Marie-Françoise; Canal, Isabelle; Firth, Andrew E; Atkins, John F; Baranov, Pavel V; Fayet, Olivier

    2014-06-01

    Programmed ribosomal -1 frameshifting is a non-standard decoding process occurring when ribosomes encounter a signal embedded in the mRNA of certain eukaryotic and prokaryotic genes. This signal has a mandatory component, the frameshift motif: it is either a Z_ZZN tetramer or a X_XXZ_ZZN heptamer (where ZZZ and XXX are three identical nucleotides) allowing cognate or near-cognate repairing to the -1 frame of the A site or A and P sites tRNAs. Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%. The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ. The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting. PMID:24875478

  2. Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli

    PubMed Central

    Singh, Dharam; Chang, Ssu-Jean; Lin, Pei-Hsun; Averina, Olga V.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2009-01-01

    Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments. PMID:19144914

  3. The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

    PubMed Central

    GUILLIER, MAUDE; ALLEMAND, FRÉDÉRIC; GRAFFE, MONIQUE; RAIBAUD, SOPHIE; DARDEL, FRÉDÉRIC; SPRINGER, MATHIAS; CHIARUTTINI, CLAUDE

    2005-01-01

    The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control. PMID:15840820

  4. The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome

    PubMed Central

    GU, SHAN-QING; PESKE, FRANK; WIEDEN, HANS-JOACHIM; RODNINA, MARINA V.; WINTERMEYER, WOLFGANG

    2003-01-01

    The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit. The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit. PMID:12702815

  5. The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome.

    PubMed

    Gu, Shan-Qing; Peske, Frank; Wieden, Hans-Joachim; Rodnina, Marina V; Wintermeyer, Wolfgang

    2003-05-01

    The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit. The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit. PMID:12702815

  6. Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7.

    PubMed Central

    Robert, F; Gagnon, M; Sans, D; Michnick, S; Brakier-Gingras, L

    2000-01-01

    Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half. The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an alpha-helical domain with a beta-ribbon extension. To gain further insights into its interaction with rRNA, we cloned the S7 gene from Escherichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence. The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes. Deletion of the N-terminal 17 residues or deletion of the B hairpins (residues 72-89) severely decreased S7 affinity for the rRNA. Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal alpha-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding. Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the alpha-helices. Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain. PMID:11105763

  7. Magnesium ions and the structure of Escherichia coli ribosomal ribonucleic acid

    PubMed Central

    Rodgers, A.

    1966-01-01

    1. The effect of removing Mg2+ from a purified high-molecular-weight (1·07×106) fraction of Escherichia coli ribosomal RNA was examined by ultracentrifugation, thermal denaturation and optical rotation. 2. At moderate I (0·1m-sodium chloride), EDTA at 2–50mm has little effect on RNA; at low I, 0·01–0·04 (with tris as counter-ion), two boundaries appear. 3. The leading boundary, S20,w about 20s, is identified with the original material with counter-ion Mg2+ (`ionic atmosphere') removed, leading to an expanded form. 4. The slow boundary, 15–16s, is associated with a further loss of Mg2+ and a further expansion, sensitive to EDTA concentration: it is proposed that this Mg2+ is localized on the polynucleotide chain, i.e. `site-bound'. 5. I is important and the EDTA effect at low I is reversible if Na+ is added immediately after the EDTA: this Na+ reversibility is lost on standing at 0°. It is suggested that changes in the tertiary structure may be associated with this loss of reversibility. 6. Thermal-denaturation studies show that there is no loss of secondary structure associated with these changes: change in the optical-rotatory-dispersion spectrum in the region of the Cotton effect may be associated with this change in tertiary structure. PMID:4960869

  8. Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

    PubMed

    Weitzmann, C J; Cunningham, P R; Ofengand, J

    1990-06-25

    The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed. PMID:2194163

  9. Role of Ribosome Release in Regulation of tna Operon Expression in Escherichia coli

    PubMed Central

    Konan, Kouacou Vincent; Yanofsky, Charles

    1999-01-01

    Expression of the degradative tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In cultures growing in the absence of added tryptophan, transcription of the structural genes of the tna operon is limited by Rho-dependent transcription termination in the leader region of the operon. Tryptophan induction prevents this Rho-dependent termination, and requires in-frame translation of a 24-residue leader peptide coding region, tnaC, that contains a single, crucial, Trp codon. Studies with a lacZ reporter construct lacking the spacer region between tnaC and the first major structural gene, tnaA, suggested that tryptophan induction might involve cis action by the TnaC leader peptide on the ribosome translating the tnaC coding region. The leader peptide was hypothesized to inhibit ribosome release at the tnaC stop codon, thereby blocking Rho’s access to the transcript. Regulatory studies with deletion constructs of the tna operon of Proteus vulgaris supported this interpretation. In the present study the putative role of the tnaC stop codon in tna operon regulation in E. coli was examined further by replacing the natural tnaC stop codon, UGA, with UAG or UAA in a tnaC-stop codon-tnaA′-′lacZ reporter construct. Basal level expression was reduced to 20 and 50% when the UGA stop codon was replaced by UAG or UAA, respectively, consistent with the finding that in E. coli translation terminates more efficiently at UAG and UAA than at UGA. Tryptophan induction was observed in strains with any of the stop codons. However, when UAG or UAA replaced UGA, the induced level of expression was also reduced to 15 and 50% of that obtained with UGA as the tnaC stop codon, respectively. Introduction of a mutant allele encoding a temperature-sensitive release factor 1, prfA1, increased basal level expression 60-fold when the tnaC stop codon was UAG and 3-fold when this stop codon was UAA; basal level

  10. Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli

    PubMed Central

    Vesper, Oliver; Amitai, Shahar; Belitsky, Maria; Byrgazov, Konstantin; Kaberdina, Anna Chao; Engelberg-Kulka, Hanna; Moll, Isabella

    2016-01-01

    Summary Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3′ terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli. PMID:21944167

  11. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  12. Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli.

    PubMed

    Luidalepp, Hannes; Berger, Stefan; Joss, Oliver; Tenson, Tanel; Polacek, Norbert

    2016-05-22

    Stationary-phase bacterial cells are characterized by vastly reduced metabolic activities yielding a dormant-like phenotype. Several hibernation programs ensure the establishment and maintenance of this resting growth state. Some of the stationary phase-specific modulations affect the ribosome and its translational activity directly. In stationary-phase Escherichia coli, we observed the appearance of a 16S rRNA fragmentation event at the tip of helix 6 within the small ribosomal subunit (30S). Stationary-phase 30S subunits showed markedly reduced activities in protein biosynthesis. On the other hand, the functional performance of stationary-phase large ribosomal subunits (50S) was indistinguishable from particles isolated from exponentially growing cells. Introduction of the 16S rRNA cut in vitro at helix 6 of exponential phase 30S subunits renders them less efficient in protein biosynthesis. This indicates that the helix 6 fragmentation is necessary and sufficient to attenuate translational activities of 30S ribosomal subunits. These results suggest that stationary phase-specific cleavage of 16S rRNA within the 30S subunit is an efficient means to reduce global translation activities under non-proliferating growth conditions. PMID:27067112

  13. Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

    PubMed Central

    Tapprich, W E; Goss, D J; Dahlberg, A E

    1989-01-01

    A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis. PMID:2662189

  14. Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation.

    PubMed Central

    Nitta, I; Ueda, T; Watanabe, K

    1998-01-01

    Experimental results are presented suggesting that 23S rRNA is directly involved in the peptide bond formation usually performed on the ribosome. Although several reports have indicated that the eubacterial peptidyltransferase reaction does not necessarily require all the ribosomal proteins, the reconstitution of peptidyltransferase activity by a naked 23S rRNA without the help of any of the ribosomal proteins has not been reported previously. It is demonstrated that an E. coli 23S rRNA transcript synthesized by T7 RNA polymerase in vitro was able to promote peptide bond formation in the presence of 0.5% SDS. The reaction was inhibited by the peptidyltransferase-specific antibiotics chloramphenicol and carbomycin, and by digestion with RNases A and T1. Site-directed mutageneses at two highly conserved regions close to the peptidyltransferase center ring, G2252 to U2252 and C2507G2581 to U2507A2581, also suppressed peptide bond formation. These findings strongly suggest that 23S rRNA is the peptidyltransferase itself. PMID:9510328

  15. Structural and functional analysis of Escherichia coli ribosomes containing small deletions around position 1760 in the 23S ribosomal RNA.

    PubMed Central

    Zweib, C; Dahlberg, A E

    1984-01-01

    Three different small deletions were produced at a single Pvu 2 restriction site in E. coli 23S rDNA of plasmid pKK 3535 using exonuclease Bal 31. The deletions were located around position 1760 in 23S rRNA and were characterized by DNA sequencing as well as by direct fingerprinting and S1-mapping of the rRNA. Two of the mutant plasmids, Pvu 2-32 and Pvu 2-33, greatly reduced the growth rate of transformed cells while the third mutant, Pvu 2-14 grew as fast as cells containing the wild-type plasmid pKK 3535. All three mutant 23S rRNAs were incorporated into 50S-like particles and were even found in 70S ribosomes and polysomes in vivo. The conformation of mutant 23S rRNA in 50S subunits was probed with a double-strand specific RNase from cobra venom. These analyses revealed changes in the accessibility of cleavage sites near the deletions around position 1760 and in the area around position 800 in all three mutant rRNAs. We suggest, that an altered conformation of the rRNAs at the site of the deletion is responsible for the slow growth of cells containing mutant plasmids Pvu 2-32 and Pvu 2-33. Images PMID:6091057

  16. Depletion of Free 30S Ribosomal Subunits in Escherichia coli by Expression of RNA Containing Shine-Dalgarno-Like Sequences

    PubMed Central

    Mawn, Mary V.; Fournier, Maurille J.; Tirrell, David A.; Mason, Thomas L.

    2002-01-01

    We have constructed synthetic coding sequences for the expression of poly(α,l-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5′-GAGGAGG-3′) that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA. PMID:11751827

  17. A three-dimensional model of domain III of the Escherichia coli small ribosomal subunit.

    PubMed

    Elson, D; Spitnik-Elson, P

    1987-09-01

    A three-dimensional model of domain III (nucleotides 920 to 1395) of the 30S ribosomal subunit of E. coli is proposed. The data used as a guide in folding the secondary structure of the RNA into a tertiary structure are four long range RNA-RNA interactions proposed by us on the basis of experiments performed in this laboratory plus two sets of data from other laboratories: protein-RNA cross-linking sites for proteins S1, S3, S7, S10 and S12, and the interprotein distances determined by neutron scattering. The model is consistent with nearly all of the published experimental findings on the structure of domain III. PMID:2450593

  18. 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…

  19. Characterization of the autophosphorylation property of HflX, a ribosome-binding GTPase from Escherichia coli.

    PubMed

    Ghosh, Aditi; Dutta, Dipak; Bandyopadhyay, Kaustav; Parrack, Pradeep

    2016-07-01

    Escherichia coli HflX belongs to the widely distributed but poorly characterized HflX family of translation factor-related GTPases that is conserved from bacteria to humans. A 426-residue polypeptide that binds 50S ribosomes and has both GTPase and ATPase activities, HflX also exhibits autophosphorylation activity. We show that HflX(C), a C-terminal fragment of HflX, has an enhanced autophosphorylation activity compared to the full-length protein. Using a chemical stability assay and thin layer chromatography, we have determined that phosphorylation occurs at a serine residue. Each of the nine serine residues of HflX(C) was mutated to alanine. It was found that all but S211A retained autophosphorylation activity, suggesting that S211, located in the P-loop, was the likely site for autophosphorylation. While the S211A mutant lacked the autophosphorylation site, it possessed strong GTP binding and GTPase activities. PMID:27398305

  20. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene.

    PubMed

    Cerretti, D P; Dean, D; Davis, G R; Bedwell, D M; Nomura, M

    1983-05-11

    The genes encoding the 52 ribosomal proteins (r-proteins) of Escherichia coli are organized into approximately 19 operons scattered throughout the chromosome. One of these, the spc operon, contains the genes for ten ribosomal proteins: L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15 (rp1N, rp1X, rp1E, rpsN, rpsH, rp1F, rp1R, rpsE, rpmD, and rp1O). We now report the entire 5.9 kb nucleotide sequence of the spc operon. DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon. It has also revealed the presence of two open reading frames past the last known gene (L15) of the spc operon. One of these corresponds to a gene (pr1A or secY) which recently has been shown by others to be involved in protein export. In addition, S1 mapping experiments indicate that a significant proportion of transcription initiated from the spc operon continues not only into the two putative genes, but also without termination into the downstream alpha r-protein operon. PMID:6222285

  1. Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA

    PubMed Central

    Robert, Francis; Brakier-Gingras, Léa

    2001-01-01

    Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3′ major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion. PMID:11160889

  2. De novo Synthesis and Assembly of rRNA into Ribosomal Subunits during Cold Acclimation in Escherichia coli.

    PubMed

    Piersimoni, Lolita; Giangrossi, Mara; Marchi, Paolo; Brandi, Anna; Gualerzi, Claudio O; Pon, Cynthia L

    2016-04-24

    During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°→10°C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation. PMID:26953262

  3. SuhB Associates with Nus Factors To Facilitate 30S Ribosome Biogenesis in Escherichia coli

    PubMed Central

    Singh, Navjot; Bubunenko, Mikhail; Smith, Carol; Abbott, David M.; Stringer, Anne M.; Shi, Ronald; Court, Donald L.

    2016-01-01

    ABSTRACT A complex of highly conserved proteins consisting of NusB, NusE, NusA, and NusG is required for robust expression of rRNA in Escherichia coli. This complex is proposed to prevent Rho-dependent transcription termination by a process known as “antitermination.” The mechanism of this antitermination in rRNA is poorly understood but requires association of NusB and NusE with a specific RNA sequence in rRNA known as BoxA. Here, we identify a novel member of the rRNA antitermination machinery: the inositol monophosphatase SuhB. We show that SuhB associates with elongating RNA polymerase (RNAP) at rRNA in a NusB-dependent manner. Although we show that SuhB is required for BoxA-mediated antitermination in a reporter system, our data indicate that the major function of the NusB/E/A/G/SuhB complex is not to prevent Rho-dependent termination of rRNA but rather to promote correct rRNA maturation. This occurs through formation of a SuhB-mediated loop between NusB/E/BoxA and RNAP/NusA/G. Thus, we have reassigned the function of these proteins at rRNA and identified another key player in this complex. PMID:26980831

  4. Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli

    PubMed Central

    Tsai, Yi-Chun; Du, Dijun; Domínguez-Malfavón, Lilianha; Dimastrogiovanni, Daniela; Cross, Jonathan; Callaghan, Anastasia J.; García-Mena, Jaime; Luisi, Ben F.

    2012-01-01

    The RNA degradosome is a multi-enzyme assembly that contributes to key processes of RNA metabolism, and it engages numerous partners in serving its varied functional roles. Small domains within the assembly recognize collectively a diverse range of macromolecules, including the core protein components, the cytoplasmic lipid membrane, mRNAs, non-coding regulatory RNAs and precursors of structured RNAs. We present evidence that the degradosome can form a stable complex with the 70S ribosome and polysomes, and we demonstrate the proximity in vivo of ribosomal proteins and the scaffold of the degradosome, RNase E. The principal interactions are mapped to two, independent, RNA-binding domains from RNase E. RhlB, the RNA helicase component of the degradosome, also contributes to ribosome binding, and this is favoured through an activating interaction with RNase E. The catalytic activity of RNase E for processing 9S RNA (the ribosomal 5S RNA precursor) is repressed in the presence of the ribosome, whereas there is little affect on the cleavage of single-stranded substrates mediated by non-coding RNA, suggestings that the enzyme retains capacity to cleave unstructured substrates when associated with the ribosome. We propose that polysomes may act as antennae that enhance the rates of capture of the limited number of degradosomes, so that they become recruited to sites of active translation to act on mRNAs as they become exposed or tagged for degradation. PMID:22923520

  5. Evidence for a role of initiation factor 3 in recycling of ribosomal complexes stalled on mRNAs in Escherichia coli

    PubMed Central

    Singh, N. S.; Das, G.; Seshadri, A.; Sangeetha, R.; Varshney, U.

    2005-01-01

    Specific interactions between ribosome recycling factor (RRF) and elongation factor-G (EFG) mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EFG are also important in peptidyl-tRNA release from stalled pre-termination complexes. Unlike the post-termination complexes (harboring deacylated tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EFG in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, (i) Inclusion of IF3 with RRF and EFG results in recycling of the pre-termination complexes; (ii) IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (iii) Transduction of infC135 (which encodes a functionally compromised IF3) in E.coli LJ14 generates a ‘synthetic severe’ phenotype; (iv) The infC135 and frr1 (containing an insertion in the RRF gene promoter) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E.coli; and (v) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E.coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E.coli. PMID:16199751

  6. Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli.

    PubMed

    Capel, M S; Kjeldgaard, M; Engelman, D M; Moore, P B

    1988-03-01

    Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit. PMID:3288761

  7. Retroregulation of the synthesis of ribosomal proteins L14 and L24 by feedback repressor S8 in Escherichia coli.

    PubMed Central

    Mattheakis, L; Vu, L; Sor, F; Nomura, M

    1989-01-01

    Previous studies on regulation of the spc operon containing genes for ribosomal proteins have shown that S8, encoded by the fifth gene of the operon in Escherichia coli, is a translational repressor and regulates the synthesis of the third gene product (L5) and distal gene products by acting at a site near the L5 mRNA translation initiation site. We have now shown that S8 also regulates the synthesis of the first and second gene products (L14 and L24) of the operon by acting at the same mRNA target site--that is, the site located distal to sites coding for L14 and L24--and that mRNA degradation is involved in this retroregulation. It was shown that single base substitutions in the target site, which abolish repression of the synthesis of L5 and L5-distal gene products by S8, also cause derepression of L14-L24 synthesis. Inhibition of L14-L24 synthesis by S8 was also shown by overproducing S8 in trans from a plasmid carrying the S8 gene under lac promoter/operator control. A strain carrying temperature-sensitive mutations in genes for polynucleotide phosphorylase and RNase II was found upon shift to nonpermissive temperature to show higher differential synthesis rates of L14-L24 (and L5) relative to those of several L5-distal spc operon gene products. We suggest that repression of distal ribosomal protein synthesis by S8 triggers nucleolytic cleavage of spc operon mRNA, followed by mRNA degradation by these 3'- to 5'- exonucleases, which is then responsible for inhibition of L14-L24 synthesis. Images PMID:2643112

  8. Synthesis and characterization of modified nucleotides in the 970 hairpin loop of Escherichia coli 16S ribosomal RNA

    PubMed Central

    Abeydeera, N. Dinuka

    2009-01-01

    The synthesis of the 6-O-DPC-2-N-methylguanosine (m2G) nucleoside and the corresponding 5′-O-DMT-2′-O-TOM-protected 6-O-DPC-2-N-methylguanosine phosphoramidite is reported [DPC, diphenyl carbamoyl; DMT, 4, 4′-dimethoxytrityl; TOM, [(triisopropylsilyl)oxy]methyl]. The availability of the phosphoramidite allows for syntheses of hairpin RNAs with site-selective incorporation of 2-N-methylguanosine modification. Four 18-nt hairpin RNA analogues representing the 970-loop region (helix 31 or h31; U960–A975) of Escherichia coli 16S rRNA were synthesized with and without modifications in the loop region. Subsequently, stabilities and conformations of the singly and doubly modified RNAs were examined and compared with the corresponding unmodified RNA. Thermodynamic parameters and circular dichroism spectra are presented for the four helix 31 RNA analogues. Surprisingly, methylations in the loop region of helix 31 slightly destabilize the hairpin, which may have subtle effects on ribosome function. The hairpin construct is suitable for future ligand-binding experiments. PMID:19628400

  9. Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli

    PubMed Central

    Di Pietro, Fabio; Brandi, Anna; Dzeladini, Nadire; Fabbretti, Attilio; Carzaniga, Thomas; Piersimoni, Lolita; Pon, Cynthia L; Giuliodori, Anna Maria

    2013-01-01

    Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock. PMID:23420694

  10. Differential scanning calorimetry of whole Escherichia coli treated with the antimicrobial peptide MSI-78 indicate a multi-hit mechanism with ribosomes as a novel target

    PubMed Central

    Brannan, Alexander M.; Whelan, William A.; Cole, Emma

    2015-01-01

    Differential Scanning Calorimetry (DSC) of intact Escherichia coli (E. coli) was used to identify non-lipidic targets of the antimicrobial peptide (AMP) MSI-78. The DSC thermograms revealed that, in addition to its known lytic properties, MSI-78 also has a striking effect on ribosomes. MSI-78’s effect on DSC scans of bacteria was similar to that of kanamycin, an antibiotic drug known to target the 30S small ribosomal subunit. An in vitro transcription/translation assay helped confirm MSI-78’s targeting of ribosomes. The scrambled version of MSI-78 also affected the ribosome peak of the DSC scans, but required greater amounts of peptide to cause a similar effect to the unscrambled peptide. Furthermore, the effect of the scrambled peptide was not specific to the ribosomes; other regions of the DSC thermogram were also affected. These results suggest that MSI-78’s effects on E. coli are at least somewhat dependent on its particular structural features, rather than a sole function of its overall charge and hydrophobicity. When considered along with earlier work detailing MSI-78’s membrane lytic properties, it appears that MSI-78 operates via a multi-hit mechanism with multiple targets. PMID:26713257

  11. Cloning, sequencing, and characterization of ribosomal protein and RNA polymerase genes from the region analogous to the alpha-operon of escherichia coli in halophilic archaea, halobacterium halobium.

    PubMed

    Sano, K; Taguchi, A; Furumoto, H; Uda, T; Itoh, T

    1999-10-14

    A determination was made of the nucleotide sequence of the 3215-bp region of a ribosomal protein gene cluster (HS13, HS4, HS11, and HeL18), RNA polymerase (RNA poly D), and tRNA genes (tRNAser and tRNAarg) of halophilic Archaea Halobacterium halobium, which is analogous to the alpha-operon of Escherichia coli (tRNAser-HS13-HS4-HS11-RNA poly D-tRNAarg-HeL18). The seven-gene string was preceded by a pseudoknot-like structure similar to the proposed S4 ribosomal protein binding site of the alpha-operon mRNA leader in E. coli. Using an inducible expression system H. halobium HS4 was produced in large amounts in E. coli, and immunoblot analysis showed the S4 to constitute a 21-kDa polypeptide component of the ribosome. Analysis of the deduced amino acids sequence revealed that the HS13, HS4, and HS11 sequences including the RNA polymerase subunit are more similar to their eukaryotic than to their bacterial counterparts. HeL18, located downstream of the gene cluster analogous to the E. coli alpha-operon (S13-S11-S4-RNA poly D-L17), was similar to both the eukaryotic (eL18) and eubacterial ribosomal protein L15 located in the spc-operon, but not to L17 positioned as the terminal gene of the bacterial alpha-operon. PMID:10527834

  12. Mutations in the Escherichia coli Ribosomal Protein L22 Selectively Suppress the Expression of a Secreted Bacterial Virulence Factor

    PubMed Central

    2013-01-01

    Mutations in the ribosomal protein L22 that impair peptide-mediated translation arrest in Escherichia coli have been shown to reduce the expression of several genes, including secA, which encodes an ATPase that drives protein export via the Sec pathway. Here, we used a comparative proteomic approach to obtain insight into the global effects of the L22(Δ82-84) mutation on gene expression and protein synthesis. While the mutation did not affect or modestly affected the level of most soluble proteins, it dramatically reduced the level of antigen 43 (Ag43), a secreted virulence factor that promotes autoaggregation. The reduced protein concentration correlated with a sharp decrease in the abundance and stability of Ag43 mRNA. We found that the overexpression of secA or the inactivation of genes that encode presecretory and membrane proteins restored Ag43 production in the L22 mutant strain. Furthermore, impairment of the Sec pathway in a wild-type strain reduced Ag43 production but did not significantly affect the synthesis of other presecretory proteins. Taken together, these results indicate that Ag43 gene expression is exquisitely sensitive to the status of the Sec machinery and strongly suggest that the L22 mutation decreases the Ag43 concentration indirectly by reducing secA expression. Our results imply the existence of a novel regulatory mechanism in which the efficiency of protein export is coupled to gene expression and help to explain the modulation of SecA synthesis that has been observed in response to secretion stress. PMID:23625843

  13. The 70 S monosome accumulation and in vitro initiation complex formation by Escherichia coli ribosomes at 5 C. Ph.D. Thesis

    NASA Technical Reports Server (NTRS)

    Broeze, R. J.; Pope, D. H.

    1978-01-01

    The inhibition of translation which is observed after shifting Escherichia coli to low temperature was investigated. 70 S ribosomes were isolated from E. coli 8 hours after a shift to 5 C synthesized protein in the absence of added mRNA (i.e., endogenous protein synthesis by 70 S monosomes) at a rate which was three times greater than the rate of endogenous protein synthesis by 70 S ribosomes which were isolated at the time of the shift to 5 C. Calculations based on the rates of endogenous protein synthesis and polyphenylalanine synthesis indicate that 70 S monosomes comprise only 0.1% of the total E. coli 70 S ribosome population after 8 hours at 5 c. Experiments designed to test initiation complex formation on ApUpG or formaldehyde treated MS-2 viral RNA demonstrated that, although the rate of formation of 30 S initiation complexes was not inhibited, the rate of formation of active 70 S initiation complexes, able to react with puromycin, was inhibited to a great extent at 5 C. A model depicting the effects of low temperature on the E. coli translation system is proposed.

  14. Analysis of the conformation of the 3' major domain of Escherichia coli16S ribosomal RNA using site-directed photoaffinity crosslinking.

    PubMed Central

    Montpetit, A; Payant, C; Nolan, J M; Brakier-Gingras, L

    1998-01-01

    The 3' major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3' major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity. PMID:9814765

  15. Chloramphenicol-induced changes in the synthesis of ribosomal, transfer, and messenger ribonucleic acids in Escherichia coli B/r.

    PubMed

    Shen, V; Bremer, H

    1977-06-01

    The synthesis of ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA) was measured in Escherichia coli B/r after the addition of 100 mug of chloramphenicol (CAM) per ml to cultures growing either in one of three minimal media (succinate, glycerol, or glucose) or in one of the same three media supplemented with 20 amino acids. (i) During CAM treatment, rRNA and tRNA were synthesized in the same relative proportions (85:15) as during exponential growth. The faster accumulation of tRNA relative to rRNA in CAM was due to a decreased stability of rRNA that is synthesized in the presence of or immediately before the addition of CAM. (ii) CAM stimulated the synthesis of rRNA and tRNA two- to eightfold. The results fell into two groups; one group was from studies done in minimal media and the other was from amino acid-supplemented media. In each group the stimulation decreased with increasing growth rate of the culture during exponential growth before the addition of CAM; however, the stimulation in minimal media was lower than that in amino acid-supplemented media. (iii) CAM caused an increase in the proportion of rRNA and tRNA synthesis and a corresponding decrease in the proportion of mRNA synthesis. In minimal media, the residual proportion of mRNA synthesis after CAM treatment was 10 to 15% of total RNA synthesis; in amino acid-supplemented media this proportion was 0 to 10%. In either case, the residual proportion of mRNA synthesis was independent of the proportions observed during exponential growth in these media. (iv) The absolute rate of mRNA synthesis decreased severalfold with the addition of CAM; i.e., the rate of synthesis of rRNA and tRNA was increased at the expense of mRNA synthesis. (v) During exponential growth, the fraction of the instantaneous rate of total RNA synthesis that corresponds to mRNA is a function of both the growth rate and the presence or absence of amino acids in the growth medium: in the absence of amino acids

  16. Conserved codon composition of ribosomal protein coding genes in Escherichia coli, Mycobacterium tuberculosis and Saccharomyces cerevisiae: lessons from supervised machine learning in functional genomics.

    PubMed

    Lin, Kui; Kuang, Yuyu; Joseph, Jeremiah S; Kolatkar, Prasanna R

    2002-06-01

    Genomics projects have resulted in a flood of sequence data. Functional annotation currently relies almost exclusively on inter-species sequence comparison and is restricted in cases of limited data from related species and widely divergent sequences with no known homologs. Here, we demonstrate that codon composition, a fusion of codon usage bias and amino acid composition signals, can accurately discriminate, in the absence of sequence homology information, cytoplasmic ribosomal protein genes from all other genes of known function in Saccharomyces cerevisiae, Escherichia coli and Mycobacterium tuberculosis using an implementation of support vector machines, SVM(light). Analysis of these codon composition signals is instructive in determining features that confer individuality to ribosomal protein genes. Each of the sets of positively charged, negatively charged and small hydrophobic residues, as well as codon bias, contribute to their distinctive codon composition profile. The representation of all these signals is sensitively detected, combined and augmented by the SVMs to perform an accurate classification. Of special mention is an obvious outlier, yeast gene RPL22B, highly homologous to RPL22A but employing very different codon usage, perhaps indicating a non-ribosomal function. Finally, we propose that codon composition be used in combination with other attributes in gene/protein classification by supervised machine learning algorithms. PMID:12034849

  17. Use of molecular beacons to probe for messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage codons in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Gao, Wenwu; Tyagi, Sanjay; Kramer, Fred R.; Goldman, Emanuel

    2000-03-01

    In `5'-translational blockage,' significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low-usage codons prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes, monosomes and ribosomes-free. New hybridization probes, called `molecular beacons,' and standard slot-blots, were used to detect test messages containing either consecutive low-usage AGG (arginine) or synonymous high-usage CGU insertions near the 5' end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present compared to high-usage codons. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted, or when the arginine codons were inserted near the 3' end. These data indicate that at least some mRNA is released from ribosomes during 5'-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.

  18. Escherichia coli (E. coli)

    MedlinePlus

    ... so you might hear about E. coli being found in drinking water, which are not themselves harmful, but indicate the ... at CDC Foodborne disease Travelers' Health: Safe Food & Water Healthy Swimming E. coli Infection & Farm ... Word file Microsoft Excel file Audio/Video file Apple ...

  19. Incorporation of single dinitrophenyl-modified proteins in to the 30S subunit of Escherichia coli ribosomes by total reconstitution for localization by immune electron microscopy

    SciTech Connect

    Olah, T.V.

    1989-01-01

    The ribosome is a structurally defined organelle whose function is central to the existence of all organisms. It is the unique site of protein biosynthesis in all cells. A detailed understanding of ribosome structure is essential in understanding the process of translation. This thesis represents a new approach to the systematic localization of individual proteins contained in the small subunit of Escherichia coli ribosomes using immunoelectron microscopy. All 30S proteins were purified using high performance liquid chromatography (HPLC) and eight isolated proteins (S12,S21,S14,S19,S18,S17,S16 and S13) were derivatized with 2,4-(3,5-{sup 3}H)dinitrofluorobenzene (DNFB). The extent of modification of these proteins was estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214nm to detect protein and 360nm (to detect dinitrophenyl groups). Each dinitrophenylated protein was introduced in place of the corresponding unmodified protein into totally reconstituted 30S subunits. Antibodies raised against the DNP-hapten bound effectively to such reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. Incorporation of any of the DNP-modified proteins, with the exception of DNP-S18, does not interfere with the functionality of the ribosome as measure by the binding of Phe-tRNA{sup Phe} or the synthesis of poly(Phe) in a poly(U)-dependent manner. Results show that unmodified protein competes with DNP-protein and that DNP-protein can function, as its native counterpart, in stimulating uptake of specific proteins during reconstitution. This data provides evidence that each DNP-protein occupies the same position in 30S subunits as does the corresponding unmodified protein.

  20. A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis

    PubMed Central

    Bharat, Amrita; Blanchard, Jan E.; Brown, Eric D.

    2014-01-01

    The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counter-screens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis. PMID:23606650

  1. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation.

    PubMed

    Lawrence, Marlon G; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M; Lindahl, Lasse

    2016-07-01

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the ONLY: sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlA(crb) pause peptide. PMID:27257065

  2. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation

    PubMed Central

    Lawrence, Marlon G.; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M.; Lindahl, Lasse

    2016-01-01

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlAcrb pause peptide. PMID:27257065

  3. Effect of an uncE ribosome-binding site mutation on the synthesis and assembly of the Escherichia coli proton-translocating ATPase.

    PubMed

    Solomon, K A; Brusilow, W S

    1988-04-15

    Plasmid pRPG54, which carries the genes for the eight subunits of the proton-translocating ATPase of Escherichia coli, has been found to carry a single base change of a G to an A in the ribosome-binding site for uncE, the gene which codes for the N,N'-dicyclohexylcarbodiimide-binding subunit c of the Fo. This noncoding region mutation both lowers expression of uncE by a factor of 2-3 and affects the function of the ATPase, specifically of the Fo sector. The presence of the mutation results in a decrease in the proton permeability of the Fo or of the entire F1Fo-ATPase complex when either is synthesized from genes on a multicopy plasmid. Expression of uncE from an F1Fo plasmid carrying the wild type ribosome binding site results in increased membrane proton permeability and decreased ability of the resultant ATPase to couple a transmembrane proton gradient to ATP synthesis both in vitro and in vivo. Also, although an Fo plasmid carrying the correct ribosome-binding site causes harmful, F1-dependent proton permeability in unc+ cells (Brusilow, W. S. S. (1987) J. Bacteriol. 169, 4984-4990), an identical plasmid carrying the mutation does not, even though it still codes for a functional reconstitutable Fo. The results show a relationship between the relative level of expression of uncE from a multicopy plasmid and the assembly pathway, proton permeability, and energy-coupling characteristics of the ATPase. PMID:2895768

  4. 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

  5. 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...

  6. 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...

  7. The 16S rRNA binding site of Thermus thermophilus ribosomal protein S15: comparison with Escherichia coli S15, minimum site and structure.

    PubMed

    Serganov, A A; Masquida, B; Westhof, E; Cachia, C; Portier, C; Garber, M; Ehresmann, B; Ehresmann, C

    1996-11-01

    Binding of Escherichia coli and Thermus thermophilus ribosomal proteins S15 to a 16S ribosomal RNA fragment from T. thermophilus (nt 559-753) has been investigated in detail by extensive deletion analysis, filter-binding assays, gel mobility shift, structure probing, footprinting with chemical, enzymatic, and hydroxyl radical probes. Both S15 proteins recognize two distinct sites. The first one maps in the bottom of helix 638-655/717-734 (H22) and in the three-way junction between helix 560-570/737-747 (H20), helix 571-600/606-634 (H21), and H22. The second is located in a conserved purine-rich region in the center of H22. The first site provides a higher contribution to the free energy of binding than the second one, and both are required for efficient binding. A short RNA fragment of 56 nt containing these elements binds S15 with high affinity. The structure of the rRNA is constrained by the three-way junction and requires both magnesium and S15 to be stabilized. A 3D model, derived by computer modeling with the use of experimental data, suggests that the bound form adopts a Y-shaped conformation, with a quasi-coaxial stacking of H22 on H20, and H21 forming an acute angle with H22. In this model, S15 binds to the shallow groove of the RNA on the exterior side of the Y-shaped structure, making contact with the two sites, which are separated by one helix turn. PMID:8903343

  8. Methylation sites in Escherichia coli ribosomal RNA: localization and identification of four new sites of methylation in 23S rRNA.

    PubMed

    Smith, J E; Cooperman, B S; Mitchell, P

    1992-11-10

    Four previously undetermined sites of methylation are mapped in Escherichia coli 23S rRNA employing a novel combination of methods. First, using a double-isotope approach, the total number of methyl groups in 23S rRNA was determined to be 14.9 +/- 1.6. Second, hybridization of methyl-labeled rRNA to complementary DNA restriction fragments and PAGE analysis were used to purify RNA-DNA heteroduplexes and to quantify methyl groups within specific 23S rRNA fragments. Third, the methylated nucleosides in these fragments were identified and quantified using HPLC, confirming the presence of 14 methylation sites in 23S rRNA, four more than had been previously identified. In contrast, a similar set of analyses conducted on 16S rRNA gave evidence for 10 sites of methylation, at all approximate locations consistent with published 16S methylated nucleoside identities and locations. Selected regions of the 23S rRNA molecule containing previously unidentified methylated nucleosides were released by site-directed cleavage with ribonuclease H and isolated by PAGE. Sites of methylation within the RNA fragments were determined by classical oligonucleotide analyses. The four newly identified methylation sites in 23S rRNA are m2G-1835, m5C-1962, m6A-2503, and m2G at one of positions 2445-2447. Together with previously described sites of modification, these new sites form a group that is clustered in a current model for the three-dimensional organization of the 23S rRNA in the 50S ribosomal subunit, at a locus congruent with nucleotides previously implicated in ribosomal function. PMID:1384701

  9. Pathogenic Escherichia coli.

    PubMed

    Kaper, James B; Nataro, James P; Mobley, Harry L

    2004-02-01

    Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes. PMID:15040260

  10. Effects of base change mutations within an Escherichia coli ribosomal RNA leader region on rRNA maturation and ribosome formation

    PubMed Central

    Schäferkordt, Jan; Wagner, Rolf

    2001-01-01

    The effects of base change mutations in a highly conserved sequence (boxC) within the leader of bacterial ribosomal RNAs (rRNAs) was studied. The boxC sequence preceding the 16S rRNA structural gene constitutes part of the RNase III processing site, one of the first cleavage sites on the pathway to mature 16S rRNA. Moreover, rRNA leader sequences facilitate correct 16S rRNA folding, thereby assisting ribosomal subunit formation. Mutations in boxC cause cold sensitivity and result in 16S rRNA and 30S subunit deficiency. Strains in which all rRNA operons are replaced by mutant transcription units are viable. Thermodynamic studies by temperature gradient gel electrophoresis reveal that mutant transcripts have a different, less ordered structure. In addition, RNA secondary structure differences between mutant and wild-type transcripts were determined by chemical and enzymatic probing. Differences are found in the leader RNA sequence itself but also in structurally important regions of the mature 16S rRNA. A minor fraction of the rRNA transcripts from mutant operons is not processed by RNase III, resulting in a significantly extended precursor half-life compared to the wild-type. The boxC mutations also give rise to a new aberrant degradation product of 16S rRNA. This intermediate cannot be detected in strains lacking RNase III. Together the results indicate that the boxC sequence, although important for RNase III processing, is likely to serve additional functions by facilitating correct formation of the mature 16S rRNA structure. They also suggest that quality control steps are acting during ribosome biogenesis. PMID:11504877

  11. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  12. Tn9 and IS1 inserts in a ribosomal ribonucleic acid operon of Escherichia coli are incompletely polar.

    PubMed Central

    Brewster, J M; Morgan, E A

    1981-01-01

    Transcription is known to be coupled to translation in many or all bacterial operons which code for proteins. In these operons, nonsense codons which prevent normal translation often result in premature termination of transcription (polarity). However, efficient transcription of ribosomal ribonucleic acid operons (rrn operons) occurs, although rrn transcripts are not translated. It therefore seemed possible that insertion sequences and transposable elements which are polar in protein-coding operons might not be polar in rrn operons. Previously, it has been shown (E. A. Morgan, Cell 21:257-265, 1980) that Tn10 is incompletely polar in the rrnX operon. Here we show that the transposon Tn9 and the insertion sequence IS1 also incompletely polar in rrnX. In normal cells expression of sequences distal to the insertions can be detected by genetic methods. In ultraviolet-irradiated cells expression of distal sequences is about 80% of that observed in uninterrupted rrnX operons. These observations provide evidence that ribonucleic acid polymerase molecules beginning at rrnX promoters can read through Tn9 and IS1 and that, at least in ultraviolet-irradiated cells, read-through is very efficient. Images PMID:6171559

  13. Evidence for the ability of L10 ribosomal proteins of Salmonella typhimurium and Klebsiella pneumoniae to regulate rplJL gene expression in Escherichia coli.

    PubMed

    Paton, E B; Woodmaska, M I; Kroupskaya, I V; Zhyvoloup, A N; Matsuka, G Kh

    1990-06-01

    Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae. PMID:2194828

  14. Recurrent Escherichia coli bacteremia.

    PubMed Central

    Maslow, J N; Mulligan, M E; Arbeit, R D

    1994-01-01

    Escherichia coli is the most common gram-negative organism associated with bacteremia. While recurrent E. coli urinary tract infections are well-described, recurrent E. coli bacteremia appears to be uncommon, with no episodes noted in multiple series of patients with gram-negative bacteremias. We report on 5 patients with recurrent bloodstream infections identified from a series of 163 patients with E. coli bacteremia. For each patient, the isolates from each episode were analyzed by pulsed-field gel electrophoresis (PFGE) and ribotyping and for the presence of E. coli virulence factors. For each of four patients, the index and recurrent episodes of bacteremia represented the same strain as defined by PFGE, and the strains were found to carry one or more virulence factors. The remaining patient, with two episodes of bloodstream infection separated by a 4-year interval, was infected with two isolates that did not carry any virulence factors and that were clonally related by ribotype analysis but differed by PFGE. All five patients had either a local host defense defect (three patients) or impaired systemic defenses (one patient) or both (one patient). Thus, recurrent E. coli bacteremia is likely to represent a multifactorial process that occurs in patients with impaired host defenses who are infected with virulent isolates. Images PMID:7910828

  15. deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2.

    PubMed Central

    Toone, W M; Rudd, K E; Friesen, J D

    1991-01-01

    We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli. Images PMID:2045359

  16. Selective translation during stress in Escherichia coli

    PubMed Central

    Moll, Isabella; Engelberg-Kulka, Hanna

    2016-01-01

    The bacterial stress response, a strategy to cope with environmental changes, is generally known to operate on the transcriptional level. Here, we discuss a novel paradigm for stress adaptation at the post-transcriptional level, based on the recent discovery of a stress-induced modified form of the translation machinery in Escherichia coli that is generated by MazF, the toxin component of the toxin–antitoxin (TA) module mazEF. Under stress, the induced endoribonuclease MazF removes the 3′-terminal 43 nucleotides of the 16S rRNA of ribosomes and, concomitantly, the 5′-untranslated regions (UTRs) of specific transcripts. This elegant mechanism enables selective translation due to the complementary effect of MazF on ribosomes and mRNAs, and also represents the first example of functional ribosome heterogeneity based on rRNA alteration. PMID:22939840

  17. Structure of the DNA distal to the gene for ribosomal protein S20 in Escherichia coli K12: presence of a strong terminator and an IS1 element.

    PubMed Central

    Mackie, G A

    1986-01-01

    The sequence of nucleotides extending over 2.3 kb distal to the gene for ribosomal protein S20 of E. coli has been determined. Included in the sequence is an efficient rho-independent terminator 50 b.p. distal to the coding sequence for S20, a complete copy of IS1 which lacks, however, flanking direct repeats, and finally, an open reading frame capable of encoding a 28 kDa polypeptide of unknown function. Several lines of evidence suggest that the IS1 sequence described here must represent one of the copies resident in the bacterial chromosome rather than a newly transposed copy. Northern blotting experiments show that the gene for S20 is functionally monocistronic under all conditions tested in several genetic backgrounds. Thus it seems unlikely that the distal copy of IS1 plays any role in the termination or stability of mRNA transcribed from the gene for S20. Images PMID:2429258

  18. Enterohemorrhagic Escherichia coli Adhesins.

    PubMed

    McWilliams, Brian D; Torres, Alfredo G

    2014-06-01

    Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbria) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this article have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics. PMID:26103974

  19. Emerging Enteropathogenic Escherichia coli Strains?

    PubMed Central

    Irino, Kinue; Girão, Dennys M.; Girão, Valéria B.C.; Guth, Beatriz E.C.; Vaz, Tânia M.I.; Moreira, Fabiana C.; Chinarelli, Silvia H.; Vieira, Mônica A.M.

    2004-01-01

    Escherichia coli strains of nonenteropathogenic serogroups carrying eae but lacking the enteropathogenic E. coli adherence factor plasmid and Shiga toxin DNA probe sequences were isolated from patients (children, adults, and AIDS patients) with and without diarrhea in Brazil. Although diverse in phenotype and genotype, some strains are potentially diarrheagenic. PMID:15504277

  20. EXTRAINTESTINAL PATHOGENIC ESCHERICHIA COLI (EXPEC)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Extraintestinal pathogenic Escherichia coli (ExPEC) possess virulence traits that allow them to invade, colonize, and induce disease in bodily sites outside of the gastrointestinal tract. Human diseases caused by ExPEC include urinary tract infections, neonatal meningitis, sepsis, pneumonia, surgic...

  1. Specific recognition of rpsO mRNA and 16S rRNA by Escherichia coli ribosomal protein S15 relies on both mimicry and site differentiation

    PubMed Central

    Mathy, Nathalie; Pellegrini, Olivier; Serganov, Alexander; Patel, Dinshaw J.; Ehresmann, Chantal; Portier, Claude

    2015-01-01

    Summary The ribosomal protein S15 binds to 16S rRNA, during ribosome assembly, and to its own mRNA (rpsO mRNA), affecting autocontrol of its expression. In both cases, the RNA binding site is bipartite with a common subsite consisting of a G•U/G-C motif. The second subsite is located in a three-way junction in 16S rRNA and in the distal part of a stem forming a pseudoknot in Escherichia coli rpsO mRNA. To determine the extent of mimicry between these two RNA targets, we determined which amino acids interact with rpsO mRNA. A plasmid carrying rpsO (the S15 gene) was mutagenized and introduced into a strain lacking S15 and harbouring an rpsO–lacZ translational fusion. Analysis of deregulated mutants shows that each subsite of rpsO mRNA is recognized by a set of amino acids known to interact with 16S rRNA. In addition to the G•U/G-C motif, which is recognized by the same amino acids in both targets, the other subsite interacts with amino acids also involved in contacts with helix H22 of 16S rRNA, in the region adjacent to the three-way junction. However, specific S15–rpsO mRNA interactions can also be found, probably with A(−46) in loop L1 of the pseudoknot, demonstrating that mimicry between the two targets is limited. PMID:15101974

  2. Initiation factor IF 2 binds to the alpha-sarcin loop and helix 89 of Escherichia coli 23S ribosomal RNA.

    PubMed Central

    La Teana, A; Gualerzi, C O; Dahlberg, A E

    2001-01-01

    During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2. PMID:11497435

  3. Shiga Toxin Producing Escherichia coli.

    PubMed

    Bryan, Allen; Youngster, Ilan; McAdam, Alexander J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) is among the common causes of foodborne gastroenteritis. STEC is defined by the production of specific toxins, but within this pathotype there is a diverse group of organisms. This diversity has important consequences for understanding the pathogenesis of the organism, as well as for selecting the optimum strategy for diagnostic testing in the clinical laboratory. This review includes discussions of the mechanisms of pathogenesis, the range of manifestations of infection, and the several different methods of laboratory detection of Shiga toxin-producing E coli. PMID:26004641

  4. Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

    PubMed Central

    Lancaster, Lorna E; Savelsbergh, Andreas; Kleanthous, Colin; Wintermeyer, Wolfgang; Rodnina, Marina V

    2008-01-01

    The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon–anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon–recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA–mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin. PMID:18485067

  5. Structural studies of E. coli ribosomes by spectroscopic techniques: A specialized review

    NASA Astrophysics Data System (ADS)

    Bonicontro, Adalberto; Risuleo, Gianfranco

    2005-12-01

    We present a review on our interdisciplinary line of research based on strategies of molecular biology and biophysics. These have been applied to the study of the prokaryotic ribosome of the bacterium Escherichia coli. Our investigations on this organelle have continued for more than a decade and we have adopted different spectroscopic biophysical techniques such as: dielectric and fluorescence spectroscopy as well as light scattering (photon correlation spectroscopy). Here we report studies on the whole 70S ribosomes and on the separated subunits 30S and 50S. Our results evidence intrinsic structural features of the subunits: the small shows a more "floppy" structure, while the large one appears to be more rigid. Also, an inner "kernel" formed by the RNA/protein association is found within the ribosome. This kernel is surrounded by a ribonucleoprotein complex more exposed to the solvent. Initial analyses were done on the so called Kaldtschmit-Wittmann ribosome: more recently we have extended the studies to the "tight couple" ribosome known for its better functional performance in vitro. Data evidence a phenomenological correlation between the differential biological activity and the intrinsic structural properties of the two-ribosome species. Finally, investigations were also conducted on particles treated at sub-denaturing temperatures and on ribosomes partially deproteinized by salt treatment (ribosomal cores). Results suggest that the thermal treatment and the selective removal of proteins cause analogous structural alterations.

  6. Cotranscription of two genes necessary for ribosomal protein L11 methylation (prmA) and pantothenate transport (panF) in Escherichia coli K-12.

    PubMed

    Vanet, A; Plumbridge, J A; Alix, J H

    1993-11-01

    Genetic complementation and enzyme assays have shown that the DNA region between panF, which encodes pantothenate permease, and orf1, the first gene of the fis operon, encodes prmA, the genetic determinant for the ribosomal protein L11 methyltransferase. Sequencing of this region identified one long open reading frame that encodes a protein of 31,830 Da and corresponds to the prmA gene. We found, both in vivo and in vitro, that prmA is expressed from promoters located upstream of panF and thus that the panF and prmA genes constitute a bifunctional operon. We located the major 3' end of prmA transcripts 90 nucleotides downstream of the stop codon of prmA in the DNA region upstream of the fis operon, a region implicated in the control of the expression of the fis operon. Although no promoter activity was detected immediately upstream of prmA, S1 mapping detected 5' ends of mRNA in this region, implying that some mRNA processing occurs within the bicistronic panF-prmA mRNA. PMID:8226664

  7. SEROLOGICAL CROSS-REACTIONS BETWEEN ESCHERICHIA COLI 0157 AND OTHER SPECIES OF THE GENUS ESCHERICHIA

    EPA Science Inventory

    Escherichia hermannii, a sorbitol-negative species of the genus Escherichia, has been reported to be agglutinated by Escherichia coli 0157 and four sorbitol-negative species of the genus Escherichia: . hermannii (24 isolates), Escherichia fergusonii (12 isolates), Escherichia vul...

  8. A Single Missense Mutation in a Coiled-Coil Domain of Escherichia coli Ribosomal Protein S2 Confers a Thermosensitive Phenotype That Can Be Suppressed by Ribosomal Protein S1

    PubMed Central

    Aseev, Leonid V.; Chugunov, Anton O.; Efremov, Roman G.

    2013-01-01

    Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation. PMID:23104805

  9. Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

    PubMed Central

    Ehresmann, C; Moine, H; Mougel, M; Dondon, J; Grunberg-Manago, M; Ebel, J P; Ehresmann, B

    1986-01-01

    The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far. Images PMID:2425339

  10. Quantitative assessment of ribosome drop-off in E. coli.

    PubMed

    Sin, Celine; Chiarugi, Davide; Valleriani, Angelo

    2016-04-01

    Premature ribosome drop-off is one of the major errors in translation of mRNA by ribosomes. However, repeated analyses of Ribo-seq data failed to quantify its strength inE. coli Relying on a novel highly sensitive data analysis method we show that a significant rate of ribosome drop-off is measurable and can be quantified also when cells are cultured under non-stressing conditions. Moreover, we find that the drop-off rate is highly variable, depending on multiple factors. In particular, under environmental stress such as amino acid starvation or ethanol intoxication, the drop-off rate markedly increases. PMID:26935582

  11. Quantitative assessment of ribosome drop-off in E. coli

    PubMed Central

    Sin, Celine; Chiarugi, Davide; Valleriani, Angelo

    2016-01-01

    Premature ribosome drop-off is one of the major errors in translation of mRNA by ribosomes. However, repeated analyses of Ribo-seq data failed to quantify its strength in E. coli. Relying on a novel highly sensitive data analysis method we show that a significant rate of ribosome drop-off is measurable and can be quantified also when cells are cultured under non-stressing conditions. Moreover, we find that the drop-off rate is highly variable, depending on multiple factors. In particular, under environmental stress such as amino acid starvation or ethanol intoxication, the drop-off rate markedly increases. PMID:26935582

  12. Nonchemotactic Mutants of Escherichia coli

    PubMed Central

    Armstrong, John B.; Adler, Julius; Dahl, Margaret M.

    1967-01-01

    We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged. Images PMID:5335897

  13. Peptidoglycan Hydrolases of Escherichia coli

    PubMed Central

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  14. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon

    NASA Astrophysics Data System (ADS)

    Jomaa, Ahmad; Boehringer, Daniel; Leibundgut, Marc; Ban, Nenad

    2016-01-01

    Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome-nascent chain complex.

  15. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon.

    PubMed

    Jomaa, Ahmad; Boehringer, Daniel; Leibundgut, Marc; Ban, Nenad

    2016-01-01

    Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome-nascent chain complex. PMID:26804923

  16. Kasugamycin-dependent mutants of Escherichia coli.

    PubMed Central

    Dabbs, E R

    1978-01-01

    Kasugamycin-dependent mutants have been isolated from Escherichia coli B. They were obtained through mutagenesis with ethyl methane sulfonate or nitrosoguanidine in conjunction with an antibiotic underlay technique. In the case of nitrosoguanidine, dependent mutants were obtained at a frequency of about 3% of survivors growing up in the selection. In the case of ethyl methane sulfonate, the corresponding value was 1%. Nineteen mutants showing a kasugamycin-dependent phenotype were studied. In terms of response to various temperatures and antibiotic concentrations, they were very heterogeneous, although most fell into two general classes. Genetic analysis indicated that in at least some cases, the kasugamycin-dependent phenotype was the product of two mutations. Two-dimensional gel electropherograms revealed alterations in the ribosomal proteins of seven mutants. One mutant had an alteration in protein S13, and one had an alteration in protein L14. Three showed changes in protein S9. Each of two mutants had changes in two proteins, S18 and L11. Three of these mutants additionally had protein S18 occurring in a partly altered, partly unaltered form. Images PMID:363701

  17. Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

    PubMed Central

    Chiaruttini, C; Expert-Bezançon, A; Hayes, D; Ehresmann, B

    1982-01-01

    1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide. Images PMID:6760129

  18. Entropy-based mechanism of ribosome-nucleoid segregation in E. coli cells.

    PubMed

    Mondal, Jagannath; Bratton, Benjamin P; Li, Yijie; Yethiraj, Arun; Weisshaar, James C

    2011-06-01

    In Escherichia coli, ribosomes concentrate near the cylindrical wall and at the endcaps, whereas the chromosomal DNA segregates in the more centrally located nucleoid. A simple statistical model recovers the observed ribosome-nucleoid segregation remarkably well. Plectonemic DNA is represented as a hyperbranched hard-sphere polymer, and multiple ribosomes that simultaneously translate the same mRNA strand (polysomes) are represented as freely jointed chains of hard spheres. There are no attractive interactions between particles, only excluded-volume effects. At realistic DNA and ribosome concentrations, segregation arises primarily from two effects: the DNA polymer avoids walls to maximize conformational entropy, and the polysomes occupy the empty space near the walls to maximize translational entropy. In this complex system, maximizing total entropy results in spatial organization of the components. Due to coupling of mRNA to DNA through RNA polymerase, the same entropic effects should favor the placement of highly expressed genes at the interface between the nucleoid and the ribosome-rich periphery. Such a placement would enable efficient cotranscriptional translation and facile transertion of membrane proteins into the cytoplasmic membrane. Finally, in the model, monofunctional DNA polymer beads representing the tips of plectonemes preferentially locate near the cylindrical wall. This suggests that initiation of transcription may occur preferentially near the ribosome-rich periphery. PMID:21641305

  19. 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. PMID:16790938

  20. 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.

  1. 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

  2. Dihydropteridine reductase from Escherichia coli.

    PubMed Central

    Vasudevan, S G; Shaw, D C; Armarego, W L

    1988-01-01

    A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity. It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group. It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate. The pure reductase has a yellow colour and contains bound FAD. The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor. Images Fig. 2. PMID:3060113

  3. 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...

  4. 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…

  5. A DNA structural atlas for Escherichia coli.

    PubMed

    Pedersen, A G; Jensen, L J; Brunak, S; Staerfeldt, H H; Ussery, D W

    2000-06-16

    We have performed a computational analysis of DNA structural features in 18 fully sequenced prokaryotic genomes using models for DNA curvature, DNA flexibility, and DNA stability. The structural values that are computed for the Escherichia coli chromosome are significantly different from (and generally more extreme than) that expected from the nucleotide composition. To aid this analysis, we have constructed tools that plot structural measures for all positions in a long DNA sequence (e.g. an entire chromosome) in the form of color-coded wheels (http://www.cbs.dtu. dk/services/GenomeAtlas/). We find that these "structural atlases" are useful for the discovery of interesting features that may then be investigated in more depth using statistical methods. From investigation of the E. coli structural atlas, we discovered a genome-wide trend, where an extended region encompassing the terminus displays a high of level curvature, a low level of flexibility, and a low degree of helix stability. The same situation is found in the distantly related Gram-positive bacterium Bacillus subtilis, suggesting that the phenomenon is biologically relevant. Based on a search for long DNA segments where all the independent structural measures agree, we have found a set of 20 regions with identical and very extreme structural properties. Due to their strong inherent curvature, we suggest that these may function as topological domain boundaries by efficiently organizing plectonemically supercoiled DNA. Interestingly, we find that in practically all the investigated eubacterial and archaeal genomes, there is a trend for promoter DNA being more curved, less flexible, and less stable than DNA in coding regions and in intergenic DNA without promoters. This trend is present regardless of the absolute levels of the structural parameters, and we suggest that this may be related to the requirement for helix unwinding during initiation of transcription, or perhaps to the previously observed

  6. Infection strategies of enteric pathogenic Escherichia coli

    PubMed Central

    Clements, Abigail; Young, Joanna C.; Constantinou, Nicholas; Frankel, Gad

    2012-01-01

    Enteric Escherichia coli (E. coli) are both natural flora of humans and important pathogens causing significant morbidity and mortality worldwide. Traditionally enteric E. coli have been divided into 6 pathotypes, with further pathotypes often proposed. In this review we suggest expansion of the enteric E. coli into 8 pathotypes to include the emerging pathotypes of adherent invasive E. coli (AIEC) and Shiga-toxin producing enteroaggregative E. coli (STEAEC). The molecular mechanisms that allow enteric E. coli to colonize and cause disease in the human host are examined and for two of the pathotypes that express a type 3 secretion system (T3SS) we discuss the complex interplay between translocated effectors and manipulation of host cell signaling pathways that occurs during infection. PMID:22555463

  7. 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

  8. Clinical implications of enteroadherent Escherichia coli.

    PubMed

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

    2012-10-01

    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 nonintimate adherence mediated by various adhesins. These so called "enteroadherent E. coli" categories subsequently produce 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. 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

  10. Key intermolecular interactions in the E. coli 70S ribosome revealed by coarse-grained analysis.

    PubMed

    Zhang, Zhiyong; Sanbonmatsu, Karissa Y; Voth, Gregory A

    2011-10-26

    The ribosome is a very large complex that consists of many RNA and protein molecules and plays a central role in protein biosynthesis in all organisms. Extensive interactions between different molecules are critical to ribosomal functional dynamics. In this work, intermolecular interactions in the Escherichia coli 70S ribosome are investigated by coarse-grained (CG) analysis. CG models are defined to preserve dynamic domains in RNAs and proteins and to capture functional motions in the ribosome, and then the CG sites are connected by harmonic springs, and spring constants are obtained by matching the computed fluctuations to those of an all-atom molecular dynamics (MD) simulation. Those spring constants indicate how strong the interactions are between the ribosomal components, and they are in good agreement with various experimental data. Nearly all the bridges between the small and large ribosomal subunits are indicated by CG interactions with large spring constants. The head of the small subunit is very mobile because it has minimal CG interactions with the rest of the subunit; however, a large number of small subunit proteins bind to maintain the internal structure of the head. The results show a clear connection between the intermolecular interactions and the structural and functional properties of the ribosome because of the reduced complexity in domain-based CG models. The present approach also provides a useful strategy to map interactions between molecules within large biomolecular complexes since it is not straightforward to investigate these by either atomistic MD simulations or residue-based elastic network models. PMID:21910449

  11. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon

    PubMed Central

    Jomaa, Ahmad; Boehringer, Daniel; Leibundgut, Marc; Ban, Nenad

    2016-01-01

    Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome–nascent chain complex. PMID:26804923

  12. Fosfomycin Resistance in Escherichia coli, Pennsylvania, USA

    PubMed Central

    Alrowais, Hind; McElheny, Christi L.; Spychala, Caressa N.; Sastry, Sangeeta; Guo, Qinglan; Butt, Adeel A.

    2015-01-01

    Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum β-lactamase–producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described. PMID:26488485

  13. Detection of O antigens in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipopolysaccharide on the surface of Escherichia coli constitute the O antigens, which are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in host-pathogen interactions. O antigens that are responsible for antigenic specificity of the ...

  14. 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.…

  15. 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

  16. Mechanism of Sperm Immobilization by Escherichia coli

    PubMed Central

    Prabha, Vijay; Sandhu, Ravneet; Kaur, Siftjit; Kaur, Kiranjeet; Sarwal, Abha; Mavuduru, Ravimohan S.; Singh, Shravan Kumar

    2010-01-01

    Aim. To explore the influence of Escherichia coli on the motility of human spermatozoa and its possible mechanism. Methods. Highly motile preparations of spermatozoa from normozoospermic patients were coincubated with Escherichia coli for 4 hours. At 1, 2 and 4 hours of incubation, sperm motility was determined. The factor responsible for sperm immobilization without agglutination was isolated and purified from filtrates. Results. This report confirms the immobilization of spermatozoa by E. coli and demonstrates sperm immobilization factor (SIF) excreted by E. coli. Further this factor was purified by ammonium sulfate precipitation, gel permeation chromatography, and ion-exchange chromatography. Purified SIF (56 kDa) caused instant immobilization without agglutination of human spermatozoa at 800 μg/mL and death at 2.1 mg/mL. Spermatozoa incubated with SIF revealed multiple and profound alterations involving all superficial structures of spermatozoa as observed by scanning electron microscopy. Conclusion. In conclusion, these results have shown immobilization of spermatozoa by E. coli and demonstrate a factor (SIF) produced and secreted by E. coli which causes variable structural damage as probable morphological correlates of immobilization. PMID:20379358

  17. Characterization of RNA damage under oxidative stress in Escherichia coli

    PubMed Central

    Liu, Min; Gong, Xin; Alluri, Ravi Kumar; Wu, Jinhua; Sablo, Tene’; Li, Zhongwei

    2012-01-01

    We have examined the level of 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine, in RNA in Escherichia coli under normal and oxidative stress conditions. The level of 8-oxo-G in RNA rises rapidly and remains high for hours in response to hydrogen peroxide (H2O2) challenge in a dose-dependent manner. H2O2 induced elevation of 8-oxo-G content is much higher in RNA than that of 8-hydroxydeoxyguanosine (8-oxo-dG) in DNA. Under normal conditions, the 8-oxo-G level is low in RNA isolated from the ribosome and it is nearly three times higher in non-ribosomal RNAs. In contrast, 8-oxo-G generated by a short exposure to H2O2 is almost equally distributed in various RNA species, suggesting that although ribosomal RNAs are normally less oxidized, they are not protected against exogenous H2O2. Interestingly, highly folded RNA is not protected from oxidation because 8-oxo-G generated by H2O2 treatment in vitro increases to approximately the same levels in tRNA and rRNA in both native and denatured forms. Lastly, increased RNA oxidation is closely associated with cell death by oxidative stress. Our data suggests that RNA is a primary target for reactive oxygen species and RNA oxidation is part of the paradox that cells have to deal with under oxidative stress. PMID:22718628

  18. Diagnosisand Investigation of Diarrheagenic Escherichia coli.

    PubMed

    Nataro, J P; Martinez, J

    1998-01-01

    Although most Escherichia coli are harmless commensals of the human intestine, certain specific, highly-adapted E. coli strains are capable of causing urinary tract, systemic or enteric/diarrheagenic infection. Diarrheagenic E coli are divided into six distinct categories, or pathotypes, each with a distinct pathogenic scheme (Table 1). Combined, diarrheagenic E coli have emerged as perhaps the most important enteric pathogens of man. In the developing world, the E coli categories account for more cases of gastroenteiltis among infants than any other cause (1) In addition, E coli are also the most common cause of traveller's diarrhea, which afflicts more than one million travellers to the developing world annually (1). Enterohemorrhagic E coli (EHEC) are the cause of hemolytic uremic syndrome (HUS), which has become a major foodborne threat in many parts of the developed world (2). Table 1 Categories of Diarrheagenic E. coli Category Toxins Invasion Virulence plasmid Adhesin Clinical syndrome ETEC LT, ST - Many CFA/I, CFA/II, CFA/IV, others Watery diarrhea EPEC - + 60 MDa Bundle-forming pilus Watery diarrhea of infants EHEC SLT-1, SLT-2 - 60 MDa( a ) Intimin, Fimbriae( a ) Hemorrhagic colitis, HUS EAEC EAST1( a ) ? 65 MDa( a ) AAF/I, AAF/I Watery, persistent diarrhea EIEC EIET( a ) +++ 140 MDa Ipa's(?) Watery diarrhea, dysentery DAEC ? ? ? F1845( a ) Watery diarrhea ( a )Role in pathogenesis unproven. PMID:21390758

  19. Escherichia coli bacteriuria and contraceptive method.

    PubMed

    Hooton, T M; Hillier, S; Johnson, C; Roberts, P L; Stamm, W E

    1991-01-01

    We evaluated the effects of contraceptive method on the occurrence of bacteriuria and vaginal colonization with Escherichia coli in 104 women who were evaluated prior to having sexual intercourse, the morning after intercourse, and 24 hours later. After intercourse, the prevalence of E coli bacteriuria increased slightly in oral contraceptive users but dramatically in both foam and condom users and diaphragm-spermicide users. Twenty-four hours later, the prevalence of bacteriuria remained significantly elevated only in the latter two groups. Similarly, vaginal colonization with E coli was more dramatic and persistent in users of diaphragm-spermicide and foam and condoms. Vaginal colonization with Candida species, enterococci, and staphylococci also increased significantly in diaphragm-spermicide users after intercourse. We conclude that use of the diaphragm with spermicidal jelly or use of a spermicidal foam with a condom markedly alters normal vaginal flora and strongly predisposes users to the development of vaginal colonization and bacteriuria with E coli. PMID:1859519

  20. Adhesion behaviors of Escherichia coli on hydroxyapatite.

    PubMed

    Kamitakahara, Masanobu; Takahashi, Shohei; Yokoi, Taishi; Inoue, Chihiro; Ioku, Koji

    2016-04-01

    Optimum design of support materials for microorganisms is required for the construction of bioreactors. However, the effects of support materials on microorganisms are still unclear. In this study, we investigated the adhesion behavior of Escherichia coli (E. coli) on hydroxyapatite (HA), polyurethane (PU), poly(vinyl chloride) (PVC), and carbon (Carbon) to obtain basic knowledge for the design of support materials. The total metabolic activity and number of E. coli adhering on the samples followed the order of HA ≈ Carbon>PVC>PU. On the other hand, the water contact angle of the pellet surfaces followed the order of HAcoli. The results implied that HA has a potential as a support material for microorganisms used in bioreactors. PMID:26838837

  1. Escherichia coli in retail processed food.

    PubMed Central

    Pinegar, J. A.; Cooke, E. M.

    1985-01-01

    Four thousand two hundred and forty six samples of retail processed food were examined for the presence of Escherichia coli. Overall 12% of samples contained this organism, cakes and confectionery being more frequently contaminated (28%) than meat and meat based products (9%). Contamination was more frequent in the summer months than in the colder weather and 27% of the contaminated foods contained greater than 10(3) E. coli/g. E. coli from meat and meat based products were more commonly resistant to one or more antibiotics (14%) than were confectionery strains (1%). The significance of these findings in relation to the E. coli population of the human bowel is discussed. PMID:3894508

  2. Escherichia coli in retail processed food.

    PubMed

    Pinegar, J A; Cooke, E M

    1985-08-01

    Four thousand two hundred and forty six samples of retail processed food were examined for the presence of Escherichia coli. Overall 12% of samples contained this organism, cakes and confectionery being more frequently contaminated (28%) than meat and meat based products (9%). Contamination was more frequent in the summer months than in the colder weather and 27% of the contaminated foods contained greater than 10(3) E. coli/g. E. coli from meat and meat based products were more commonly resistant to one or more antibiotics (14%) than were confectionery strains (1%). The significance of these findings in relation to the E. coli population of the human bowel is discussed. PMID:3894508

  3. Vaginal Lactobacillus isolates inhibit uropathogenic Escherichia coli.

    PubMed

    Atassi, Fabrice; Brassart, Dominique; Grob, Philipp; Graf, Federico; Servin, Alain L

    2006-04-01

    The purpose of this study was to investigate the antibacterial activities of Lactobacillus jensenii KS119.1 and KS121.1, and Lactobacillus gasserii KS120.1 and KS124.3 strains isolated from the vaginal microflora of healthy women, against uropathogenic, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strains IH11128 and 7372 involved in recurrent cystitis. We observed that some of the Lactobacillus isolates inhibited the growth and decreased the viability of E. coli IH11128 and 7372. In addition, we observed that adhering Lactobacillus strains inhibited adhesion of E. coli IH11128 onto HeLa cells, and inhibited internalization of E. coli IH11128 within HeLa cells. PMID:16553843

  4. FTIR nanobiosensors for Escherichia coli detection

    PubMed Central

    Greppi, Gianfranco; Marongiu, Maria Laura; Roggero, Pier Paolo; Ravindranath, Sandeep P; Mauer, Lisa J; Schibeci, Nicoletta; Perria, Francesco; Piccinini, Massimo; Innocenzi, Plinio; Irudayaraj, Joseph

    2012-01-01

    Summary Infections due to enterohaemorrhagic E. coli (Escherichia coli) have a low incidence but can have severe and sometimes fatal health consequences, and thus represent some of the most serious diseases due to the contamination of water and food. New, fast and simple devices that monitor these pathogens are necessary to improve the safety of our food supply chain. In this work we report on mesoporous titania thin-film substrates as sensors to detect E. coli O157:H7. Titania films treated with APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde) were functionalized with specific antibodies and the absorption properties monitored. The film-based biosensors showed a detection limit for E. coli of 1 × 102 CFU/mL, constituting a simple and selective method for the effective screening of water samples. PMID:23019542

  5. Uropathogenic Escherichia coli-associated exotoxins

    PubMed Central

    Welch, Rodney A.

    2015-01-01

    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 blood stream 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. PMID:27337488

  6. Production of antibody fragments in Escherichia coli.

    PubMed

    Katsuda, Tomohisa; Sonoda, Hiroyuki; Kumada, Yoichi; Yamaji, Hideki

    2012-01-01

    Escherichia coli is a host widely used in the industrial production of recombinant proteins. However, the expression of heterologous proteins in E. coli often encounters the formation of inclusion bodies, which are insoluble and nonfunctional protein aggregates. For the successful production of antibody fragments, which includes single-chain variable fragments (scFvs), we describe here the modification of linker, signal, and Shine-Dalgarno (SD) sequences, the coexpression of cytoplasmic and periplasmic chaperones, and a method for fed-batch cultivation with exponential feed. PMID:22907360

  7. 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

  8. 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. PMID:27337488

  9. Large plasmids of avian Escherichia coli isolates.

    PubMed

    Doetkott, D M; Nolan, L K; Giddings, C W; Berryhill, D L

    1996-01-01

    The plasmid DNA of 30 Escherichia coli isolates from chickens was extracted and examined using techniques designed to isolate large plasmids. This plasmid DNA was examined for the presence of certain known virulence-related genes including cvaC, traT, and some aerobactin-related sequences. Seventeen of the 30 isolates contained from one to four plasmids greater than 50 kb in size. Eleven of these 17 strains possessed plasmids greater than 100 kb in size. Therefore, E. coli isolates of chickens frequently contain large plasmids, and many of these plasmids are likely to contain virulence-related sequences. PMID:8980827

  10. Towards a classification of E. coli ribosomal proteins: A hypothetical `small ribosome' as a primitive protein-synthesizing apparatus

    NASA Astrophysics Data System (ADS)

    Ohnishi, Koji

    1984-12-01

    Homologies were searched among the published primary sequences of 51 E. coli ribosomal proteins, partly by ‘eye’ and partly by computer-assisted methods. By employing Moore and Goodman's alignment statistics for evaluating homology levels, 33 out of these 51 ribosomal proteins has been classified into 9 homology groups, some of which being yet tentative and remaining to be further analyzed. Taking it into consideration that most ribosomal protein genes are clustered at str- stc region, rif region and several other regions, these results strongly suggest that most or all of the contemporary ribosomal proteins must have evolved by repeated gene duplications of very few (or only one) primitive ancestral ribosomal protein gene(s). Thus it is most reasonable to propose that a ‘ small ribosome’ consisting of very few (or only one) ribosomal protein(s) must have existed as a primitive protein-synthesizing apparatus.

  11. Novel compound for identifying Escherichia coli.

    PubMed Central

    Watkins, W D; Rippey, S R; Clavet, C R; Kelley-Reitz, D J; Burkhardt, W

    1988-01-01

    A new chromogenic compound, 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide, was found to be useful for the rapid, specific, differential identification of Escherichia coli in the sanitary analysis of shellfish and wastewater. Of 1,025 presumptively positive colonies (blue) and 583 presumptively negative colonies (nonblue), only 1% false-negative and 5% false-positive results were found. PMID:3046494

  12. Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In United States, it is estimated that non-O157 Shiga toxin-producing Escherichia coli (STEC) cause more illnesses than STEC O157:H7, and the majority of cases of non-O157 STEC infections is due to serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 STEC. The diseas...

  13. Draft genome sequence of Escherichia coli LCT-EC106.

    PubMed

    Li, Tianzhi; Pu, Fei; Yang, Rentao; Fang, Xiangqun; Wang, Junfeng; Guo, Yinghua; Chang, De; Su, Longxiang; Guo, Na; Jiang, Xuege; Zhao, Jiao; Liu, Changting

    2012-08-01

    Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans. Here, we present the complete genome sequence of Escherichia coli LCT-EC106, which was isolated from CGMCC 1.2385. PMID:22843582

  14. Time course of large ribosomal subunit assembly in E. coli cells overexpressing a helicase inactive DbpA protein.

    PubMed

    Gentry, Riley C; Childs, Jared J; Gevorkyan, Jirair; Gerasimova, Yulia V; Koculi, Eda

    2016-07-01

    DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways. PMID:27194011

  15. Natural plasmid transformation in Escherichia coli.

    PubMed

    Tsen, Suh-Der; Fang, Suh-Sen; Chen, Mei-Jye; Chien, Jun-Yi; Lee, Chih-Chun; Tsen, Darwin Han-Lin

    2002-01-01

    Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli. PMID:12065899

  16. Systems Metabolic Engineering of Escherichia coli.

    PubMed

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

    2016-05-01

    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. PMID:27223822

  17. 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

  18. Mechanisms of Emerging Diarrheagenic Escherichia coli Infection.

    PubMed

    Khan, Mohammed A.; Steiner, Ted S.

    2002-04-01

    Diarrheagenic Escherichia coli organisms are major causes of morbidity and mortality worldwide. Although most strains of E. coli are harmless commensals, a few types have emerged that are capable of disrupting the normal physiology of the human gut, producing illness ranging from watery diarrhea to fatal hemorrhagic colitis. Diarrheagenic E. coli cause infection by a variety of complex mechanisms, some of which are incompletely understood. These include adherence, elaboration of toxigenic mediators, invasion of the intestinal mucosa, and transportation of bacterial proteins into the host cells. Specific components of the host-microbial interaction that cause damage have been identified, increasing our understanding of the mechanisms of diarrhea. This article reviews some of the recent findings about the pathogenesis and infectious processes involved in three emerging pathotypes of this fascinating gram-negative bacterium. PMID:11927041

  19. Molecular mechanisms of Escherichia coli pathogenicity.

    PubMed

    Croxen, Matthew A; Finlay, B Brett

    2010-01-01

    Escherichia coli is a remarkable and diverse organism. This normally harmless commensal needs only to acquire a combination of mobile genetic elements to become a highly adapted pathogen capable of causing a range of diseases, from gastroenteritis to extraintestinal infections of the urinary tract, bloodstream and central nervous system. The worldwide burden of these diseases is staggering, with hundreds of millions of people affected annually. Eight E. coli pathovars have been well characterized, and each uses a large arsenal of virulence factors to subvert host cellular functions to potentiate its virulence. In this Review, we focus on the recent advances in our understanding of the different pathogenic mechanisms that are used by various E. coli pathovars and how they cause disease in humans. PMID:19966814

  20. 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.

  1. Prodigiosin - A Multifaceted Escherichia coli Antimicrobial Agent.

    PubMed

    Danevčič, Tjaša; Borić Vezjak, Maja; 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

  2. Action of sodium deoxycholate on Escherichia coli

    SciTech Connect

    D'Mello, A.; Yotis, W.W.

    1987-08-01

    Sodium deoxycholate is used in a number of bacteriological media for the isolation and classification of gram-negative bacteria from food and the environment. Initial experiments to study the effect of deoxycholate on the growth parameters of Escherichia coli showed an increase in the lag time constant and generation time and a decrease in the growth rate constant total cell yield of this microorganisms. Cell fractionation studies indicated that sodium deoxycholate at levels used in bacteriological media interferes with the incorporation of (U-/sup 14/C)glucose into the cold-trichloroacetic acid-soluble, ethanol-soluble, and trypsin-soluble cellular fractions of E. coli. Finally, sodium deoxycholate interfered with the flagellation and motility of Proteus mirabilis and E. coli. It would appear then that further improvement of the deoxycholate medium may be in order.

  3. The synthesis of ribonucleic acid during inhibition of Escherichia coli by chlortetracycline

    PubMed Central

    Holmes, Isobel A.; Wild, D. G.

    1965-01-01

    1. During inhibition of Escherichia coli by chlortetracycline, protein synthesis was sharply reduced whereas synthesis of RNA was much less affected. 2. Most of the RNA made during inhibition was contained in particles that sedimented more slowly than ribosomes. 3. The particles were more sensitive than ribosomes to degradation by ultrasonic vibrations and ribonuclease and differed from ribosomes in their behaviour during chromatography on DEAE-cellulose. 4. The particles contained two species of RNA that differed slightly in their sedimentation properties from the two RNA components found in ribosomes. 5. The nature of the events taking place during inhibition by chlortetracycline is discussed with particular reference to the status of the particles that accumulate and to the mode of action of this and other antibiotics. ImagesFig. 2. PMID:16749115

  4. Unconventional initiator tRNAs sustain Escherichia coli

    PubMed Central

    Samhita, Laasya; Shetty, Sunil; Varshney, Umesh

    2012-01-01

    Of all tRNAs, initiator tRNA is unique in its ability to start protein synthesis by directly binding the ribosomal P-site. This ability is believed to derive from the almost universal presence of three consecutive G-C base (3G-C) pairs in the anticodon stem of initiator tRNA. Consistent with the hypothesis, a plasmid-borne initiator tRNA with one, two, or all 3G-C pairs mutated displays negligible initiation activity when tested in a WT Escherichia coli cell. Given this, the occurrence of unconventional initiator tRNAs lacking the 3G-C pairs, as in some species of Mycoplasma and Rhizobium, is puzzling. We resolve the puzzle by showing that the poor activity of unconventional initiator tRNAs in E. coli is because of competition from a large pool of the endogenous WT initiator tRNA (possessing the 3G-C pairs). We show that E. coli can be sustained on an initiator tRNA lacking the first and third G-C pairs; thereby reducing the 3G-C rule to a mere middle G-C requirement. Two general inferences following from our findings, that the activity of a mutant gene product may depend on its abundance in the cell relative to that of the WT, and that promiscuous initiation with elongator tRNAs has the potential to enhance phenotypic diversity without affecting genomic integrity, have been discussed. PMID:22829667

  5. 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

  6. COMPARATIVE RESISTANCE OF ESCHERICHIA COLI AND ENTEROCOCCI TO CHLORINATION

    EPA Science Inventory

    Pure cultures of Escherichia coli and Enterococcus faecium were inactivated by free chlorine and monochloramine. ndigenous E. coli and enterococci in wastewater effluents were also inactivated. elective bacteriological media specifically designed for the enumeration of the target...

  7. Profiling of Escherichia coli Chromosome database.

    PubMed

    Yamazaki, Yukiko; Niki, Hironori; Kato, Jun-ichi

    2008-01-01

    The Profiling of Escherichia coli Chromosome (PEC) database (http://www.shigen.nig.ac.jp/ecoli/pec/) is designed to allow E. coli researchers to efficiently access information from functional genomics studies. The database contains two principal types of data: gene essentiality and a large collection of E. coli genetic research resources. The essentiality data are based on data compilation from published single-gene essentiality studies and on cell growth studies of large-deletion mutants. Using the circular and linear viewers for both whole genomes and the minimal genome, users can not only gain an overview of the genome structure but also retrieve information on contigs, gene products, mutants, deletions, and so forth. In particular, genome-wide exhaustive mutants are an essential resource for studying E. coli gene functions. Although the genomic database was constructed independently from the genetic resources database, users may seamlessly access both types of data. In addition to these data, the PEC database also provides a summary of homologous genes of other bacterial genomes and of protein structure information, with a comprehensive interface. The PEC is thus a convenient and useful platform for contemporary E. coli researchers. PMID:18392982

  8. Logarithmic Sensing in Escherichia coli Bacterial Chemotaxis

    PubMed Central

    Kalinin, Yevgeniy V.; Jiang, Lili; Tu, Yuhai; Wu, Mingming

    2009-01-01

    We studied the response of swimming Escherichia coli (E. coli) bacteria in a comprehensive set of well-controlled chemical concentration gradients using a newly developed microfluidic device and cell tracking imaging technique. In parallel, we carried out a multi-scale theoretical modeling of bacterial chemotaxis taking into account the relevant internal signaling pathway dynamics, and predicted bacterial chemotactic responses at the cellular level. By measuring the E. coli cell density profiles across the microfluidic channel at various spatial gradients of ligand concentration grad[L] and the average ligand concentration [L]¯near the peak chemotactic response region, we demonstrated unambiguously in both experiments and model simulation that the mean chemotactic drift velocity of E. coli cells increased monotonically with grad [L]/[L]¯ or ∼grad(log[L])—that is E. coli cells sense the spatial gradient of the logarithmic ligand concentration. The exact range of the log-sensing regime was determined. The agreements between the experiments and the multi-scale model simulation verify the validity of the theoretical model, and revealed that the key microscopic mechanism for logarithmic sensing in bacterial chemotaxis is the adaptation kinetics, in contrast to explanations based directly on ligand occupancy. PMID:19289068

  9. Multiscale Spatial Organization of RNA Polymerase in Escherichia coli

    PubMed Central

    Endesfelder, Ulrike; Finan, Kieran; Holden, Seamus J.; Cook, Peter R.; Kapanidis, Achillefs N.; Heilemann, Mike

    2013-01-01

    Nucleic acid synthesis is spatially organized in many organisms. In bacteria, however, the spatial distribution of transcription remains obscure, owing largely to the diffraction limit of conventional light microscopy (200–300 nm). Here, we use photoactivated localization microscopy to localize individual molecules of RNA polymerase (RNAP) in Escherichia coli with a spatial resolution of ∼40 nm. In cells growing rapidly in nutrient-rich media, we find that RNAP is organized in 2–8 bands. The band number scaled directly with cell size (and so with the chromosome number), and bands often contained clusters of >70 tightly packed RNAPs (possibly engaged on one long ribosomal RNA operon of 6000 bp) and clusters of such clusters (perhaps reflecting a structure like the eukaryotic nucleolus where many different ribosomal RNA operons are transcribed). In nutrient-poor media, RNAPs were located in only 1–2 bands; within these bands, a disproportionate number of RNAPs were found in clusters containing ∼20–50 RNAPs. Apart from their importance for bacterial transcription, our studies pave the way for molecular-level analysis of several cellular processes at the nanometer scale. PMID:23823236

  10. 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...

  11. Cyanide degradation by an Escherichia coli strain.

    PubMed

    Figueira, M M; Ciminelli, V S; de Andrade, M C; Linardi, V R

    1996-05-01

    Chemical formation of a glucose-cyanide complex was necessary for metabolic degradation of cyanide at concentrations up to 50.0 mg/L by a strain of Escherichia coli isolated from gold extraction circuit liquids. Ammonia accumulating during the culture log phase as the sole nitrogen by-product was further utilized for bacterial growth. Washed (intact) cells, harvested at different periods of bacterial growth on cyanide, consumed oxygen in presence of cyanide. These findings suggest that metabolism of cyanide involved a dioxygenase enzyme that converted cyanide directly to ammonia, without the formation of cyanate. PMID:8640610

  12. Enterotoxigenic Escherichia coli: Orchestrated host engagement.

    PubMed

    Fleckenstein, James M; Munson, George M; Rasko, David A

    2013-01-01

    The enterotoxigenic Escherichia coli are a pervasive cause of serious diarrheal illness in developing countries. Presently, there is no vaccine to prevent these infections, and many features of the basic pathogenesis of these organisms remain poorly understood. Until very recently most pathogenesis studies had focused almost exclusively on a small subset of known "classical" virulence genes, namely fimbrial colonization factors and the heat-labile (LT) and heat stable (ST) enterotoxins. However, recent investigations of pathogen-host interactions reveal a surprisingly complex and intricately orchestrated engagement involving the interplay of classical and "novel" virulence genes, as well as participation of genes highly conserved in the E. coli species. These studies may inform further rational approaches to vaccine development for these important pathogens. PMID:23892244

  13. Engineering the Escherichia coli Fermentative Metabolism

    NASA Astrophysics Data System (ADS)

    Orencio-Trejo, M.; Utrilla, J.; Fernández-Sandoval, M. T.; Huerta-Beristain, G.; Gosset, G.; Martinez, A.

    Fermentative metabolism constitutes a fundamental cellular capacity for industrial biocatalysis. Escherichia coli is an important microorganism in the field of metabolic engineering for its well-known molecular characteristics and its rapid growth. It can adapt to different growth conditions and is able to grow in the presence or absence of oxygen. Through the use of metabolic pathway engineering and bioprocessing techniques, it is possible to explore the fundamental cellular properties and to exploit its capacity to be applied as industrial biocatalysts to produce a wide array of chemicals. The objective of this chapter is to review the metabolic engineering efforts carried out with E. coli by manipulating the central carbon metabolism and fermentative pathways to obtain strains that produce metabolites with high titers, such as ethanol, alanine, lactate and succinate.

  14. 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.

  15. Gas signatures from Escherichia coli and Escherichia coli-inoculated human whole blood

    PubMed Central

    2013-01-01

    Background The gaseous headspace above naïve Escherichia Coli (E. coli) cultures and whole human blood inoculated with E. coli were collected and analyzed for the presence of trace gases that may have the potential to be used as novel, non-invasive markers of infectious disease. Methods The naïve E. coli culture, LB broth, and human whole blood or E. coli inoculated whole blood were incubated in hermetically sealable glass bioreactors at 37°C for 24 hrs. LB broth and whole human blood were used as controls for background volatile organic compounds (VOCs). The headspace gases were collected after incubation and analyzed using a gas chromatographic system with multiple column/detector combinations. Results Six VOCs were observed to be produced by E. coli-infected whole blood while there existed nearly zero to relatively negligible amounts of these gases in the whole blood alone, LB broth, or E. coli-inoculated LB broth. These VOCs included dimethyl sulfide (DMS), carbon disulfide (CS2), ethanol, acetaldehyde, methyl butanoate, and an unidentified gas S. In contrast, there were several VOCs significantly elevated in the headspace above the E. coli in LB broth, but not present in the E. coli/blood mixture. These VOCs included dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), methyl propanoate, 1-propanol, methylcyclohexane, and unidentified gases R2 and Q. Conclusions This study demonstrates 1) that cultivated E. coli in LB broth produce distinct gas profiles, 2) for the first time, the ability to modify E. coli-specific gas profiles by the addition of whole human blood, and 3) that E. coli-human whole blood interactions present different gas emission profiles that have the potential to be used as non-invasive volatile biomarkers of E. coli infection. PMID:23842518

  16. 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

  17. Escherichia coli as a bioreporter in ecotoxicology.

    PubMed

    Robbens, Johan; Dardenne, Freddy; Devriese, Lisa; De Coen, Wim; Blust, Ronny

    2010-11-01

    Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future. PMID:20803141

  18. Escherichia coli biofilm: development and therapeutic strategies.

    PubMed

    Sharma, G; Sharma, S; Sharma, P; Chandola, D; Dang, S; Gupta, S; Gabrani, R

    2016-08-01

    Escherichia coli biofilm consists of a bacterial colony embedded in a matrix of extracellular polymeric substances (EPS) which protects the microbes from adverse environmental conditions and results in infection. Besides being the major causative agent for recurrent urinary tract infections, E. coli biofilm is also responsible for indwelling medical device-related infectivity. The cell-to-cell communication within the biofilm occurs due to quorum sensors that can modulate the key biochemical players enabling the bacteria to proliferate and intensify the resultant infections. The diversity in structural components of biofilm gets compounded due to the development of antibiotic resistance, hampering its eradication. Conventionally used antimicrobial agents have a restricted range of cellular targets and limited efficacy on biofilms. This emphasizes the need to explore the alternate therapeuticals like anti-adhesion compounds, phytochemicals, nanomaterials for effective drug delivery to restrict the growth of biofilm. The current review focuses on various aspects of E. coli biofilm development and the possible therapeutic approaches for prevention and treatment of biofilm-related infections. PMID:26811181

  19. Discrepancies in the enumeration of Escherichia coli.

    PubMed

    Ray, B; Speck, M L

    1973-04-01

    Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO(4).7H(2)O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods. PMID:4572980

  20. Role of Escherichia coli in Biofuel Production.

    PubMed

    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

  1. [Population genomic researches of Escherichia coli].

    PubMed

    Wu, Y R; Yang, R F; Cui, Y J

    2016-06-01

    Population genomics, an interdiscipline of genomics and population genetics, is booming in recent years with the rapid growth number of deciphered genomes and revolutionizes the understanding of bacterial population diversity and evolution dynamics. It also largely improves the prevention and control of infectious disease through providing more accurate genotyping and source-tracing results and more comprehensive characteristics of emerging pathogens. In this review, taking one of the best characterized bacteria, Escherichia coli, as model, we reviewed the phylogenetic relationship across its five major populations (designated A, B1, B2, D and E); and summarized researches on molecular mutation rate, selection signals, and patterns of adaptive evolution. We also described the application of population genomics in responding against large-scale outbreaks of E. coli O157:H7 and E. coli O104:H4. These results indicated that, although being a novel discipline, population genomics has played an important role in deciphering bacterial population structures, exploring evolutionary patterns and combating emerging infectious diseases. PMID:27256740

  2. 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

  3. (p)ppGpp-dependent and -independent pathways for salt tolerance in Escherichia coli.

    PubMed

    Tarusawa, Takefusa; Ito, Shion; Goto, Simon; Ushida, Chisato; Muto, Akira; Himeno, Hyouta

    2016-07-01

    Addition of some kinds of translation inhibitors targeting the ribosome such as kasugamycin to the culture medium as well as removal of a ribosome maturation factor or a ribosomal protein provides Escherichia coli cells with tolerance to high salt stress. Here, we found that another kind of translation inhibitor, serine hydroxamate (SHX), which induces amino acid starvation leading to (p)ppGpp production, also has a similar effect, but via a different pathway. Unlike kasugamycin, SHX was not effective in (p)ppGpp-null mutant cells. SHX and depletion of RsgA, a ribosome maturation factor, had an additive effect on salt tolerance, while kasugamycin or depletion of RsgA did not. These results indicate the presence of two distinct pathways, (p)ppGpp-dependent and -independent pathways, for salt tolerance of E. coli cell. Both pathways operate even in the absence of σ(S), an alternative sigma factor involved in the stationary phase or stress response. Hastened activation of the exocytoplasmic stress-specific sigma factor, σ(E), after salt shock was observed in the cells treated with SHX, as has been observed in the cells treated with a translation inhibitor or depleted of a ribosome maturation factor. PMID:26823481

  4. 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...

  5. Non-O157 Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC), also known as verocytotoxin-producing E. coli, are important food-borne pathogens responsible for outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). STEC that cause HC and HUS are also referred to as enterohemorrhagic E. coli (E...

  6. Virulence Gene Regulation in Escherichia coli.

    PubMed

    Mellies, Jay L; Barron, Alex M S

    2006-01-01

    Escherichia colicauses three types of illnesses in humans: diarrhea, urinary tract infections, and meningitis in newborns. The acquisition of virulence-associated genes and the ability to properly regulate these, often horizontally transferred, loci distinguishes pathogens from the normally harmless commensal E. coli found within the human intestine. This review addresses our current understanding of virulence gene regulation in several important diarrhea-causing pathotypes, including enteropathogenic, enterohemorrhagic,enterotoxigenic, and enteroaggregativeE. coli-EPEC, EHEC, ETEC and EAEC, respectively. The intensely studied regulatory circuitry controlling virulence of uropathogenicE. coli, or UPEC, is also reviewed, as is that of MNEC, a common cause of meningitis in neonates. Specific topics covered include the regulation of initial attachment events necessary for infection, environmental cues affecting virulence gene expression, control of attaching and effacing lesionformation, and control of effector molecule expression and secretion via the type III secretion systems by EPEC and EHEC. How phage control virulence and the expression of the Stx toxins of EHEC, phase variation, quorum sensing, and posttranscriptional regulation of virulence determinants are also addressed. A number of important virulence regulators are described, including the AraC-like molecules PerA of EPEC, CfaR and Rns of ETEC, and AggR of EAEC;the Ler protein of EPEC and EHEC;RfaH of UPEC;and the H-NS molecule that acts to silence gene expression. The regulatory circuitry controlling virulence of these greatly varied E. colipathotypes is complex, but common themes offerinsight into the signals and regulators necessary forE. coli disease progression. PMID:26443571

  7. Molecular characterization and clonal relationships among Escherichia coli strains isolated from broiler chickens with colisepticemia.

    PubMed

    Barbieri, Nicolle Lima; de Oliveira, Aline Luísa; Tejkowski, Thiago Moreira; Pavanelo, Daniel Brisotto; Matter, Letícia Beatriz; Pinheiro, Sandra Regina Schincariol; Vaz, Tânia Mara Ibelli; Nolan, Lisa K; Logue, Catherine M; de Brito, Benito Guimarães; Horn, Fabiana

    2015-01-01

    This study characterized 52 Escherichia coli isolates from distinct diseased organs of 29 broiler chickens with clinical symptoms of colibacillosis in the Southern Brazilian state of Rio Grande do Sul. Thirty-eight isolates were highly virulent and 14 were virtually avirulent in 1-day-old chicks, yet all isolates harbored virulence factors characteristic of avian pathogenic E. coli (APEC), including those related to adhesion, iron acquisition, and serum resistance. E. coli reference collection phylogenetic typing showed that isolates belonged mostly to group D (39%), followed by group A (29%), group B1 (17%), and group B2 (15%). Phylogenetic analyses using the Amplified Ribosomal DNA Restriction Analysis and pulse-field gel electrophoresis methods were used to discriminate among isolates displaying the same serotype, revealing that five birds were infected with two distinct APEC strains. Among the 52 avian isolates, 2 were members of the pandemic E. coli O25:H4-B2-ST131 clone. PMID:25514382

  8. Efficient production of indigoidine in Escherichia coli.

    PubMed

    Xu, Fuchao; Gage, David; Zhan, Jixun

    2015-08-01

    Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor L-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l L-glutamine. Given the relatively high price of L-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of L-glutamine and subsequent indigoidine production. Among the four tested salts including (NH4)2SO4, NH4Cl, (NH4)2HPO4 and KNO3, (NH4)2HPO4 showed the best effect on improving the titer of indigoidine. Different concentrations of (NH4)2HPO4 were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH4)2HPO4. This work provides two efficient methods for the production of this promising blue pigment in E. coli. PMID:26109508

  9. Phosphoglucomutase Mutants of Escherichia coli K-12

    PubMed Central

    Adhya, Sankar; Schwartz, Maxime

    1971-01-01

    Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accumulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-1-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glucose-1-phosphate is also strongly depressed, since amino acid- or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm mutations isolated map at about 16 min on the genetic map, between purE and the gal operon. PMID:4942754

  10. Structure of common pili from Escherichia coli.

    PubMed Central

    McMichael, J C; Ou, J T

    1979-01-01

    Several important properties of the common pili from Escherichia coli are discussed. These pili were resistant to the gentle Folin-Ciocalteau reagent methods for protein detection and were not readily solubilized by sodium dodecyl sulfate. They were found to contain a reducing sugar but not peptidoglycan. The pilin had multiple conformations in sodium dodecyl sulfate solution, and the appearance of multiple bands on sodium dodecyl sulfate gels did not necessarily indicate heterogeneity of the preparation. The ilus subunit was found to be a different protein than outer membrane III, which has the same apparent molecular weight. In addition, we conformed the results of Brinton (Trans. N.Y. Acad. Sci 27:1003-1054, 1965): that there is a dramatic change in the properties of pili after they are heated at pH values below 2. Images PMID:37233

  11. Novel antigens for enterotoxigenic Escherichia coli vaccines.

    PubMed

    Fleckenstein, James; Sheikh, Alaullah; Qadri, Firdausi

    2014-05-01

    Enterotoxigenic Escherichia coli (ETEC) are the most common bacterial pathogens causing diarrhea in developing countries where they lead to hundreds of thousands of deaths, mostly in children. These organisms are a leading cause of diarrheal illness in travelers to endemic countries. ETEC pathogenesis, and consequently vaccine approaches, have largely focused on plasmid-encoded enterotoxins or fimbrial colonization factors. To date these approaches have not yielded a broadly protective vaccine. However, recent studies suggest that ETEC pathogenesis is more complex than previously appreciated and involves additional plasmid and chromosomally encoded virulence molecules that can be targeted in vaccines. Here, we review recent novel antigen discovery efforts, potential contribution of these proteins to the molecular pathogenesis of ETEC and protective immunity, and the potential implications for development of next generation vaccines for important pathogens. These proteins may help to improve the effectiveness of future vaccines by making them simpler and possibly broadly protective because of their conserved nature. PMID:24702311

  12. Escherichia coli photoreactivating enzyme: purification and properties

    SciTech Connect

    Snapka, R.M.; Sutherland, B.M.

    1980-01-01

    Researchers have purified large quantities of Escherichia coli photoreactivating enzyme to apparent homogeneity and have studied its physical and chemical properties. The enzyme has a molecular weight of 36,800 and a S/sub 20,w//sup 0/ of 3.72 S. Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids. The N terminus is arginine. The purified enzyme contained up to 13% carbohydrate by weight. The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine. The enzyme is also associated with RNA containing uracil, adenine, guanine, and cytosine with no unusual bases detected.

  13. Glucose-lactose diauxie in Escherichia coli.

    PubMed

    Loomis, W F; Magasanik, B

    1967-04-01

    Growth of Escherichia coli in medium containing glucose, at a concentration insufficient to support full growth, and containing lactose, is diauxic. A mutation in the gene, CR, which determines catabolite repression specific to the lac operon, was found to relieve glucose-lactose but not glucose-maltose diauxie. Furthermore, a high concentration of lactose was shown to overcome diauxie in a CR(+) strain. Studies on the induction of beta-galactosidase by lactose suggested that glucose inhibits induction by 10(-2)m lactose. Preinduction of the lac operon was found to overcome this effect. The ability of glucose to prevent expression of the lac operon by reducing the internal concentration of inducer as well as by catabolite repression is discussed. PMID:5340309

  14. Direct Upstream Motility in Escherichia coli

    PubMed Central

    Kaya, Tolga; Koser, Hur

    2012-01-01

    We provide an experimental demonstration of positive rheotaxis (rapid and continuous upstream motility) in wild-type Escherichia coli freely swimming over a surface. This hydrodynamic phenomenon is dominant below a critical shear rate and robust against Brownian motion and cell tumbling. We deduce that individual bacteria entering a flow system can rapidly migrate upstream (>20 μm/s) much faster than a gradually advancing biofilm. Given a bacterial population with a distribution of sizes and swim speeds, local shear rate near the surface determines the dominant hydrodynamic mode for motility, i.e., circular or random trajectories for low shear rates, positive rheotaxis for moderate flow, and sideways swimming at higher shear rates. Faster swimmers can move upstream more rapidly and at higher shear rates, as expected. Interestingly, we also find on average that both swim speed and upstream motility are independent of cell aspect ratio. PMID:22500751

  15. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed

    Adler, H; Mural, R; Suttle, B

    1992-04-01

    Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media. PMID:1551829

  16. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed Central

    Adler, H; Mural, R; Suttle, B

    1992-01-01

    Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media. Images PMID:1551829

  17. Genetic Analysis of an Escherichia coli Syndrome

    PubMed Central

    Lennette, Evelyne T.; Apirion, David

    1971-01-01

    A mutant strain of Escherichia coli that fails to recover from prolonged (72 hr) starvation also fails to grow at 43 C. Extracts of this mutant strain show an increased ribonuclease II activity as compared to extracts of the parental strain, and stable ribonucleic acid is degraded to a larger extent in this strain during starvation. Ts+ transductants and revertants were tested for all the above-mentioned phenotypes. All the Ts+ transductants and revertants tested behaved like the Ts+ parental strain, which suggests that all the observed phenotypes are caused by a single sts (starvation-temperature sensitivity) mutation. The reversion rate from sts− to sts+ is rather low but is within the range of reversion rates for other single-site mutations. Three-point transduction crosses located this sts mutation between the ilv and rbs genes. The properties of sts+/sts− merozygotes suggested that the Ts− phenotype of this mutation is recessive. PMID:4945197

  18. Genes under positive selection in Escherichia coli

    PubMed Central

    Petersen, Lise; Bollback, Jonathan P.; Dimmic, Matt; Hubisz, Melissa; Nielsen, Rasmus

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome, including cell surface proteins such as beta barrel porins, presumably because of the involvement of these genes in evolutionary arms races with other bacteria, phages, and/or the host immune system. Structural mapping of positively selected sites on trans-membrane beta barrel porins reveals that the residues under positive selection occur almost exclusively in the extracellular region of the proteins that are enriched with sites known to be targets of phages, colicins, or the host immune system. More surprisingly, we also find a number of other categories of genes that show very strong evidence for positive selection, such as the enigmatic rhs elements and transposases. Based on structural evidence, we hypothesize that the selection acting on transposases is related to the genomic conflict between transposable elements and the host genome. PMID:17675366

  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. gltBDF operon of Escherichia coli.

    PubMed Central

    Castaño, I; Bastarrachea, F; Covarrubias, A A

    1988-01-01

    A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega. Images PMID:2448295

  1. Thiol-sensitive genes of Escherichia coli.

    PubMed Central

    Javor, G T

    1989-01-01

    The effect of 1-thioglycerol on the expression of genes of Escherichia coli was investigated. Pulse-labeled proteins from aerobically growing, 1-thioglycerol-treated E. coli were separated by two-dimensional gel electrophoresis, and their radioactivities were compared with those of identical proteins from nontreated cells. The first 10 min of exposure to thiol stimulated the synthesis of 10% of the observed proteins and inhibited the production of 16% of the proteins. After 30 min of growth with thiol, the synthesis of 44% of the observed proteins was inhibited and synthesis of 18% of the proteins was stimulated. In general, the expression of genes of carbohydrate metabolism, amino acid metabolism, and protein biosynthesis were inhibited, while nucleic acid synthetic and repair gene expressions showed mixed responses. Synthesis of transport proteins was not affected. Transient stimulation of oxidative-stress proteins and sustained stimulation of the expressions of trxB, ompA, and ompB genes and those of several unidentified gene products were also observed. Whether these complex responses merely reflect adjustments by cellular subsystems to a suddenly reducing environment or whether they are manifestations of a reductive-stress regulon will have to await genetic analysis of this phenomenon. Images PMID:2676982

  2. 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

  3. Escherichia coli mutants deficient in exonuclease VII.

    PubMed Central

    Chase, J W; Richardson, C C

    1977-01-01

    Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains. Images PMID:320198

  4. The spatial biology of transcription and translation in rapidly growing Escherichia coli

    PubMed Central

    Bakshi, Somenath; Choi, Heejun; Weisshaar, James C.

    2015-01-01

    Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing Escherichia coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0–3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20–30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription

  5. A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

    PubMed Central

    Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry; Potter, Clinton S; Carragher, Bridget; Williamson, James R

    2014-01-01

    Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3′ domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3′-domain is unanchored and the 5′-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells. DOI: http://dx.doi.org/10.7554/eLife.04491.001 PMID:25313868

  6. Protein folding in the cell envelope of Escherichia coli.

    PubMed

    De Geyter, Jozefien; Tsirigotaki, Alexandra; Orfanoudaki, Georgia; Zorzini, Valentina; Economou, Anastassios; Karamanou, Spyridoula

    2016-01-01

    While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines. PMID:27573113

  7. An Integrated System for Precise Genome Modification in Escherichia coli.

    PubMed

    Tas, Huseyin; Nguyen, Cac T; Patel, Ravish; Kim, Neil H; Kuhlman, Thomas E

    2015-01-01

    We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome. Genome changes are introduced first through the integration of a 1.3 kbp Landing Pad consisting of a gene conferring resistance to tetracycline (tetA) or the ability to metabolize the sugar galactose (galK). The Landing Pad is then excised as a result of double-strand breaks by the homing endonuclease I-SceI, and replaced with DNA fragments bearing the desired change via λ-Red mediated homologous recombination. Repair of the double strand breaks and counterselection against the Landing Pad (using NiCl2 for tetA or 2-deoxy-galactose for galK) allows the isolation of modified bacteria without the use of additional antibiotic selection. We demonstrate the power of this method to make a variety of genome modifications: the exact integration, without any extraneous sequence, of the lac operon (~6.5 kbp) to any desired location in the genome and without the integration of antibiotic markers; the scarless deletion of ribosomal rrn operons (~6 kbp) through either intrachromosomal or oligonucleotide recombination; and the in situ fusion of native genes to fluorescent reporter genes without additional perturbation. PMID:26332675

  8. An Integrated System for Precise Genome Modification in Escherichia coli

    PubMed Central

    Tas, Huseyin; Nguyen, Cac T.; Patel, Ravish; Kim, Neil H.; Kuhlman, Thomas E.

    2015-01-01

    We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome. Genome changes are introduced first through the integration of a 1.3 kbp Landing Pad consisting of a gene conferring resistance to tetracycline (tetA) or the ability to metabolize the sugar galactose (galK). The Landing Pad is then excised as a result of double-strand breaks by the homing endonuclease I-SceI, and replaced with DNA fragments bearing the desired change via λ-Red mediated homologous recombination. Repair of the double strand breaks and counterselection against the Landing Pad (using NiCl2 for tetA or 2-deoxy-galactose for galK) allows the isolation of modified bacteria without the use of additional antibiotic selection. We demonstrate the power of this method to make a variety of genome modifications: the exact integration, without any extraneous sequence, of the lac operon (~6.5 kbp) to any desired location in the genome and without the integration of antibiotic markers; the scarless deletion of ribosomal rrn operons (~6 kbp) through either intrachromosomal or oligonucleotide recombination; and the in situ fusion of native genes to fluorescent reporter genes without additional perturbation. PMID:26332675

  9. Nucleotide sequence corresponding to five chemotaxis genes in Escherichia coli.

    PubMed Central

    Mutoh, N; Simon, M I

    1986-01-01

    The nucleotide sequence of DNA which contains five chemotaxis-related genes of Escherichia coli, cheW, cheR, cheB, cheY, and cheZ, and part of the cheA gene was determined. Molecular weights of the polypeptides encoded by these genes were calculated from translated amino acid sequences, and they were 18,100 for cheW, 32,700 for cheR, 37,500 for cheB, 14,100 for cheY, and 24,000 for cheZ. Nucleotide sequences which could act as ribosome-binding sites were found in the upstream region of each gene. After the termination codon of the cheW gene, a typical rho-independent transcription termination signal was observed. There are no other open reading frames long enough to encode polypeptides in this region except those which code for the two previously reported genes tar and tap. PMID:3510184

  10. Escherichia coli in chronic inflammatory bowel diseases: An update on adherent invasive Escherichia coli pathogenicity

    PubMed Central

    Martinez-Medina, Margarita; Garcia-Gil, Librado Jesus

    2014-01-01

    Escherichia coli (E. coli), and particularly the adherent invasive E. coli (AIEC) pathotype, has been increasingly implicated in the ethiopathogenesis of Crohn’s disease (CD). E. coli strains with similar pathogenic features to AIEC have been associated with other intestinal disorders such as ulcerative colitis, colorectal cancer, and coeliac disease, but AIEC prevalence in these diseases remains largely unexplored. Since AIEC was described one decade ago, substantial progress has been made in deciphering its mechanisms of pathogenicity. However, the molecular bases that characterize the phenotypic properties of this pathotype are still not well resolved. A review of studies focused on E. coli populations in inflammatory bowel disease (IBD) is presented here and we discuss about the putative role of this species on each IBD subtype. Given the relevance of AIEC in CD pathogenesis, we present the latest research findings concerning AIEC host-microbe interactions and pathogenicity. We also review the existing data regarding the prevalence and abundance of AIEC in CD and its association with other intestinal diseases from humans and animals, in order to discuss the AIEC disease- and host-specificity. Finally, we highlight the fact that dietary components frequently found in industrialized countries may enhance AIEC colonization in the gut, which merits further investigation and the implementation of preventative measures. PMID:25133024

  11. 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

  12. 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...

  13. Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli*

    PubMed Central

    Fan, Haitian; Hahm, Joseph; Diggs, Stephen; Perry, J. Jefferson P.; Blaha, Gregor

    2015-01-01

    The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3′, 5′-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes to small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response. PMID:26163516

  14. Structure of the E. coli ribosome-EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM.

    PubMed

    Fischer, Niels; Neumann, Piotr; Konevega, Andrey L; Bock, Lars V; Ficner, Ralf; Rodnina, Marina V; Stark, Holger

    2015-04-23

    Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3 Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8 Å), but provides more detailed information (2.65 Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes. PMID:25707802

  15. Meta-Analysis of Transcriptional Responses to Mastitis-Causing Escherichia coli

    PubMed Central

    Younis, Sidra; Javed, Qamar; Blumenberg, Miroslav

    2016-01-01

    Bovine mastitis is a widespread disease in dairy cows, and is often caused by bacterial mammary gland infection. Mastitis causes reduced milk production and leads to excessive use of antibiotics. We present meta-analysis of transcriptional profiles of bovine mastitis from 10 studies and 307 microarrays, allowing identification of much larger sets of affected genes than any individual study. Combining multiple studies provides insight into the molecular effects of Escherichia coli infection in vivo and uncovers differences between the consequences of E. coli vs. Staphylococcus aureus infection of primary mammary epithelial cells (PMECs). In udders, live E. coli elicits inflammatory and immune defenses through numerous cytokines and chemokines. Importantly, E. coli infection causes downregulation of genes encoding lipid biosynthesis enzymes that are involved in milk production. Additionally, host metabolism is generally suppressed. Finally, defensins and bacteria-recognition genes are upregulated, while the expression of the extracellular matrix protein transcripts is silenced. In PMECs, heat-inactivated E. coli elicits expression of ribosomal, cytoskeletal and angiogenic signaling genes, and causes suppression of the cell cycle and energy production genes. We hypothesize that heat-inactivated E. coli may have prophylactic effects against mastitis. Heat-inactivated S. aureus promotes stronger inflammatory and immune defenses than E. coli. Lipopolysaccharide by itself induces MHC antigen presentation components, an effect not seen in response to E. coli bacteria. These results provide the basis for strategies to prevent and treat mastitis and may lead to the reduction in the use of antibiotics. PMID:26933871

  16. 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

  17. The N-degradome of Escherichia coli

    PubMed Central

    Humbard, Matthew A.; Surkov, Serhiy; De Donatis, Gian Marco; Jenkins, Lisa M.; Maurizi, Michael R.

    2013-01-01

    The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates. PMID:23960079

  18. Biocontrol of Escherichia coli O157

    PubMed Central

    Boyacioglu, Olcay; Sharma, Manan; Sulakvelidze, Alexander; Goktepe, Ipek

    2013-01-01

    The effect of a bacteriophage cocktail (EcoShield™) that is specific against Escherichia coli O157:H7 was evaluated against a nalidixic acid-resistant enterohemorrhagic E. coli O157:H7 RM4407 (EHEC) strain on leafy greens stored under either (1) ambient air or (2) modified atmosphere (MA; 5% O2/35% CO2/60% N2). Pieces (~2 × 2 cm2) of leafy greens (lettuce and spinach) inoculated with 4.5 log CFU/cm2 EHEC were sprayed with EcoShield™ (6.5 log PFU/cm2). Samples were stored at 4 or 10°C for up to 15 d. On spinach, the level of EHEC declined by 2.38 and 2.49 log CFU/cm2 at 4 and 10°C, respectively, 30 min after phage application (p ≤ 0.05). EcoShield™ was also effective in reducing EHEC on the surface of green leaf lettuce stored at 4°C by 2.49 and 3.28 log units in 30 min and 2 h, respectively (p ≤ 0.05). At 4°C under atmospheric air, the phage cocktail significantly (p ≤ 0.05) lowered the EHEC counts in one day by 1.19, 3.21 and 3.25 log CFU/cm2 on spinach, green leaf and romaine lettuce, respectively compared with control (no bacteriophage) treatments. When stored under MA at 4°C, phages reduced (p ≤ 0.05) EHEC populations by 2.18, 3.50 and 3.13 log CFU/cm2, on spinach, green leaf and romaine lettuce. At 10°C, EHEC reductions under atmospheric air storage were 1.99, 3.90 and 3.99 log CFU/cm2 (p ≤ 0.05), while population reductions under MA were 3.08, 3.89 and 4.34 logs on spinach, green leaf and romaine lettuce, respectively, compared with controls (p ≤ 0.05). The results of this study showed that bacteriophages were effective in reducing the levels of E. coli O157:H7 on fresh leafy produce, and that the reduction was further improved when produce was stored under the MA conditions. PMID:23819107

  19. Effect of the "RNA control" locus in Escherichia coli on RNA bacteriophage R23 replication.

    PubMed Central

    Ernberg, J; Sköld, O

    1976-01-01

    The effect of the rel gene of Escherichia coli on the RNA synthesis induced by phage R23 was studied. This RNA phage has the property of inhibiting ribosomal RNA formation and completely dominating the RNA synthesis of the host. Phage-specific RNA formation was found to be dependent on the allelic state of the rel gene. Determinations of RNA synthesis were made by both cumulative and short-term incorporations of uracil and adenine. Variations in labeling of nucleotide pools were compensated for by determining specific activities of ATP and UTP and using these values to obtain true, relative rates of RNA synthesis. PMID:768516

  20. Energetics of glycylglycine transport in Escherichia coli.

    PubMed

    Cowell, J L

    1974-10-01

    The transport system for glycylglycine in Escherichia coli behaves like a shock-sensitive transport system. The initial rate of transport is reduced 85% by subjecting whole cells to osmotic shock, and glycylglycine is not transported by membrane vesicles. The energetics of transport was studied with strain ML 308-225 and its mutant DL-54, which is deficient in Ca(2+)- and Mg(2+)-stimulated adenosine 5'-triphosphatase (EC 3.6.1.3) activity. It is concluded that active transport of glycylglycine, like other shock-sensitive transport systems, has an obligatory requirement for phosphate bond energy, but not for respiration or the energized state of the membrane. The major evidence for this conclusion is as follows. (i) Uptake of glycylglycine is severely inhibited by arsenate. (ii) Oxidizable energy sources such as d-lactate, succinate, and ascorbate, which is mediated by N-methylphenazinium methylsulfate, cannot serve as energy sources for the transport of glycylglycine in DL-54, which lacks oxidative phosphorylation. (iii) When energy is supplied only from adenosine-5'-triphosphate produced by glycolysis (anaerobic transport assays with glucose as the energy source in DL-54), substantial uptake of glycylglycine is observed. (iv) When the Ca(2+)-Mg(2+)-adenosine triphosphatase activity is absent but substrate-level phosphorylations and electron transport are operating (glucose as the energy source in DL-54), transport of glycylglycine shows significant resistance to the uncouplers, dinitrophenol and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. PMID:4278690

  1. 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.

  2. Colonization factors of enterotoxigenic Escherichia coli.

    PubMed

    Madhavan, T P Vipin; Sakellaris, Harry

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a major cause of life-threatening diarrheal disease around the world. The major aspects of ETEC virulence are colonization of the small intestine and the secretion of enterotoxins which elicit diarrhea. Intestinal colonization is mediated, in part, by adhesins displayed on the bacterial cell surface. As colonization of the intestine is the critical first step in the establishment of an infection, it represents a potential point of intervention for the prevention of infections. Therefore, colonization factors (CFs) have been important subjects of research in the field of ETEC virulence. Research in this field has revealed that ETEC possesses a large array of serologically distinct CFs that differ in composition, structure, and function. Most ETEC CFs are pili (fimbriae) or related fibrous structures, while other adhesins are simple outer membrane proteins lacking any macromolecular structure. This chapter reviews the genetics, structure, function, and regulation of ETEC CFs and how such studies have contributed to our understanding of ETEC virulence and opened up potential opportunities for the development of preventive and therapeutic interventions. PMID:25596032

  3. 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

  4. Routes for fructose utilization by Escherichia coli.

    PubMed

    Kornberg, H L

    2001-07-01

    There are three main routes for the utilization of fructose by Escherichia coli. One (Route A) predominates in the growth of wild-type strains. It involves the functioning of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and a fructose operon, mapping at min. 48.7, containing genes for a membrane-spanning protein (fruA), a 1-phosphofructose kinase (fruK) and a diphosphoryl transfer protein (fruB), under negative regulation by a fruR gene mapping at min. 1.9. A second route (Route B) also involves the PTS and membrane-spanning proteins that recognize a variety of sugars possessing the 3,4,5-D-arabino-hexoseconfiguration but with primary specificity for mannose(manXYZ), mannitol (mtlA) and glucitol (gutA) and which, if over-produced, can transport also fructose. A third route (Route C), functioning in mutants devoid of Routes A and B, does not involve the PTS: fructose diffuses into the cell via an isoform (PtsG-F) of the major glucose permease of the PTS and is then phosphorylated by ATP and a manno(fructo)kinase (Mak+) specified by a normally cryptic 1032 bp ORF (yajF) of hitherto unknown function (Mak-o), mapping at min. 8.8 and corresponding to a peptide of 344 amino acids. Conversion of the Mak-o to the Mak+ phenotypeinvolves an A24D mutation in a putative regulatory region. PMID:11361065

  5. 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.

  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. The Escherichia coli divisome: born to divide.

    PubMed

    Natale, Paolo; Pazos, Manuel; Vicente, Miguel

    2013-12-01

    Septation in Escherichia coli involves complex molecular mechanisms that contribute to the accuracy of bacterial division. The proto-ring, a complex made up by the FtsZ, FtsA and ZipA proteins, forms at the beginning of the process and directs the assembly of the full divisome. Central to this complex is the FtsZ protein, a GTPase able to assemble into a ring-like structure that responds to several modulatory inputs including mechanisms to position the septum at midcell. The connection with the cell wall synthesising machinery stabilizes the constriction of the cytoplasmic membrane. Although a substantial amount of evidence supports this description, many details on how individual divisome elements are structured or how they function are subjected to controversial interpretations. We discuss these discrepancies arising from incomplete data and from technical difficulties imposed by the small size of bacteria. Future work, including more powerful imaging and reconstruction technologies, will help to clarify the missing details on the architecture and function of the bacterial division machinery. PMID:23962168

  8. Incomplete flagellar structures in Escherichia coli mutants.

    PubMed Central

    Suzuki, T; Komeda, Y

    1981-01-01

    Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative precursors of the basal body were detected in mutants with defects in flaM, flaU, flaV, and flaY. No structures homologous to the flagellar basal body or its parts were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI, flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbA, flbB, and flbD. One flaZ mutant had an incomplete flagellar basal body structure and another formed no significant structure, suggesting that flaZ is responsible for both basal body assembly and the transcription of the hag gene. Images PMID:7007337

  9. 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.

  10. Molecular Serotyping of Escherichia coli O111:H8

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate Escherichia coli serotyping is critical for pathogen diagnosis and surveillance of non-O157 shiga-toxigenic strains, however, few laboratories have this capacity. The molecular serotyping protocol described in this paper targets the somatic and flagellar antigens of E. coli O111:H8 used in...

  11. Properties and Transport Behavior among 12 Different Environmental Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli is a commonly used indicator organism for detecting the presence of fecal-borne pathogenic microorganisms in water supplies. The importance of E. coli as an indicator organism has led to numerous studies looking at cell properties and transport behavior of this microorganism. In man...

  12. Complete Draft Genome Sequence of Escherichia coli JF733.

    PubMed

    Kleiner, Gabriele R M; Wibberg, Daniel; Winkler, Anika; Kalinowski, Jörn; Wertz, John E; Friehs, Karl

    2016-01-01

    ITALIC! Escherichia coliJF733 is a strain with a long history in research on membrane proteins and processes. However, tracing back the strain development raises some questions concerning the correct genotype of JF733. Here, we present the complete draft genome of ITALIC! E. coliJF733 in order to resolve any remaining uncertainties. PMID:27103723

  13. Complete Genome Sequence of Enterotoxigenic Escherichia coli Myophage Murica

    PubMed Central

    Wilder, Joseph N.; Lancaster, Jacob C.; Cahill, Jesse L.; Rasche, Eric S.

    2015-01-01

    Murica is an rv5-like myophage that infects enterotoxigenic Escherichia coli. Pathogenic E. coli strains are responsible for many intestinal diseases, and phages that infect these bacteria may prove useful in preventing severe health issues. The following is a report of the complete genome sequence of Murica and its important features. PMID:26430048

  14. Complete Genome Sequence of Enterotoxigenic Escherichia coli Myophage Murica.

    PubMed

    Wilder, Joseph N; Lancaster, Jacob C; Cahill, Jesse L; Rasche, Eric S; Kuty Everett, Gabriel F

    2015-01-01

    Murica is an rv5-like myophage that infects enterotoxigenic Escherichia coli. Pathogenic E. coli strains are responsible for many intestinal diseases, and phages that infect these bacteria may prove useful in preventing severe health issues. The following is a report of the complete genome sequence of Murica and its important features. PMID:26430048

  15. Complete Draft Genome Sequence of Escherichia coli JF733

    PubMed Central

    Kleiner, Gabriele R. M.; Wibberg, Daniel; Winkler, Anika; Wertz, John E.; Friehs, Karl

    2016-01-01

    Escherichia coli JF733 is a strain with a long history in research on membrane proteins and processes. However, tracing back the strain development raises some questions concerning the correct genotype of JF733. Here, we present the complete draft genome of E. coli JF733 in order to resolve any remaining uncertainties. PMID:27103723

  16. EFFECT OF MANURE ON ESCHERICHIA COLI ATTACHMENT TO SOIL FRACTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli are commonly used as indicators of fecal contamination in the environment. Attachment of bacteria to soil and sediment is an important retardation factor of bacterial transport with runoff water. Despite the fact that E. coli are derived exclusively from feces/manure, the effect of ...

  17. Draft Genome Sequence of Uropathogenic Escherichia coli Strain NB8.

    PubMed

    Weng, Xing-Bei; 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

  18. Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli.

    PubMed Central

    Boylan, M; Smyth, C J; Scott, J R

    1988-01-01

    The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues. PMID:2903130

  19. 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)....

  20. Clonal relationships among bloodstream isolates of Escherichia coli.

    PubMed Central

    Maslow, J N; Whittam, T S; Gilks, C F; Wilson, R A; Mulligan, M E; Adams, K S; Arbeit, R D

    1995-01-01

    The clonal relationships among 187 bloodstream isolates of Escherichia coli from 179 patients at Boston, Mass., Long Beach, Calif., and Nairobi, Kenya, were determined by multilocus enzyme electrophoresis (MLEE), analysis of polymorphisms associated with the ribosomal operon (ribotyping), and serotyping. MLEE based on 20 enzymes resolved 101 electrophoretic types (ETs), forming five clusters; ribotyping resolved 56 distinct patterns concordant with the analysis by MLEE. The isolates at each study site formed a genetically diverse group and demonstrated similar clonal structures, with the same small subset of lineages accounting for the majority of isolates at each site. Moreover, two ribotypes accounted for approximately 30% of the isolates at each study site. One cluster contained the majority (65%) of isolates and, by direct comparison of the ETs and ribotypes of individual isolates, was genetically indistinguishable from the largest cluster for each of two other collections of E. coli causing pyelonephritis and neonatal meningitis (R. K. Selander, T. K. Korhonen, V. Väisänen-Rhen, P. H. Williams, P. E. Pattison, and D. A. Caugent, Infect. Immun. 52:213-222, 1986; M. Arthur, C. E. Johnson, R. H. Rubin, R. D. Arbeit, C. Campanelli, C. Kim, S. Steinbach, M. Agarwal, R. Wilkinson, and R. Goldstein, Infect. Immun. 57:303-313, 1989), thus defining a virulent set of lineages. The isolates within these virulent lineages typically carried DNA homologous to the adhesin operon pap or sfa and the hemolysin operon hly and expressed O1, O2, O4, O6, O18, O25, or O75 antigens. DNA homologous to pap was distributed among isolates of each major cluster, whereas hly was restricted to isolates of two clusters, typically detected in pap-positive strains, and sfa was restricted to isolates of one cluster, typically detected in pap- and hly-positive strains. The occurrence of pap-positive isolates in the same geographically and genetically divergent lineages suggests that this

  1. A Novel Cell-Based Method to Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7 is a leading cause of foodborne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stxs and inhibitors of Stx activity. A Vero...

  2. Inactivation of Escherichia coli O157:H7 and Escherichia coli 8739 in apple juice by pulsed electric fields.

    PubMed

    Evrendilek, G A; Zhang, Q H; Richter, E R

    1999-07-01

    The effect of high voltage pulsed electric field (PEF) treatment on Escherichia coli O157:H7 and generic E. coli 8739 in apple juice was investigated. Fresh apple juice samples inoculated with E. coli O157:H7 and E. coli 8739 were treated by PEF with selected parameters including electric field strength, treatment time, and treatment temperature. Samples were exposed to bipolar pulses with electric field strengths of 30, 26, 22, and 18 kV/cm and total treatment times of 172, 144, 115, and 86 micros. A 5-log reduction in both cultures was determined by a standard nonselective medium spread plate laboratory procedure. Treatment temperature was kept below 35 degrees C. Results showed no difference in the sensitivities of E. coli O157:H7 and E. coli 8739 against PEF treatment. PEF is a promising technology for the inactivation of E. coli O157:H7 and E. coli 8739 in apple juice. PMID:10419274

  3. 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

  4. Systematic Mutagenesis of the Escherichia coli Genome†

    PubMed Central

    Kang, Yisheng; Durfee, Tim; Glasner, Jeremy D.; Qiu, Yu; Frisch, David; Winterberg, Kelly M.; Blattner, Frederick R.

    2004-01-01

    A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the λRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition. PMID:15262929

  5. Routes of quinolone permeation in Escherichia coli.

    PubMed Central

    Chapman, J S; Georgopapadakou, N H

    1988-01-01

    The uptake of quinolone antibiotics by Escherichia coli was investigated by using fleroxacin (RO 23-6240, AM 833) as a prototype compound. The uptake of fleroxacin was reduced and its MIC was increased in the presence of magnesium. Quinolones induced lipopolysaccharide release, increased cell-surface hydrophobicity and outer membrane permeability to B-lactams, and sensitized cells to lysis by detergents. These effects were also antagonized by magnesium and were very similar to those seen with EDTA and gentamicin. MICs of quinolones in portin-deficient strains were increased relative to those of the parent strain, consistent with a porin pathway of entry. However, MICs were further increased in the presence of magnesium; the size of the additional increase showed a positive correlation with quinolone hydrophobicity in an OmpF- OmpC- OmpA- strain. When quinolones were mixed with divalent cations in solution, changes in quinolone fluorescence suggestive of metal chelation were observed. The addition of fleroxacin to a cell suspension resulted in a rapid initial association of fluorescence with cells, followed by a brief decrease and a final time-dependent linear increase in cell-associated fluorescence. We interpret these results as representing chelation of outer membrane-bound magnesium by fleroxacin and other quinolones, dissociation of the quinolone-magnesium complex from the outer membrane, and diffusion of the quinolone through both porins and exposed lipid domains on the outer membrane. For a given quinolone, the contribution of the porin and nonporin pathways to total uptake is influenced by the hydrophobicity of the quinolone. PMID:3132091

  6. Physiological state of Escherichia coli BJ4 growing in the large intestines of streptomycin-treated mice.

    PubMed Central

    Poulsen, L K; Licht, T R; Rang, C; Krogfelt, K A; Molin, S

    1995-01-01

    Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static. PMID:7592332

  7. 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. PMID:17057960

  8. Functions that protect Escherichia coli from DNA-protein crosslinks.

    PubMed

    Krasich, Rachel; Wu, Sunny Yang; Kuo, H Kenny; Kreuzer, Kenneth N

    2015-04-01

    Pathways for tolerating and repairing DNA-protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies (recA, recBC, recG, and uvrD), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification (dinG, ftsK, xerD, dnaJ, hflC, miaA, mnmE, mnmG, and ssrA). Inactivation of SbcCD, which can cleave DNA at protein-DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA, mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear. PMID:25731940

  9. Functions that protect Escherichia coli from DNA-protein crosslinks

    PubMed Central

    Krasich, Rachel; Wu, Sunny Yang; Kuo, H. Kenny; Kreuzer, Kenneth N

    2015-01-01

    Pathways for tolerating and repairing DNA-protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies (recA, recBC, recG, and uvrD), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification (dinG, ftsK, xerD, dnaJ, hflC, miaA, mnmE, mnmG, and ssrA). Inactivation of SbcCD, which can cleave DNA at protein-DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA, mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear. PMID:25731940

  10. 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.

  11. Serological cross-reactions between Escherichia coli O157 and other species of the genus Escherichia.

    PubMed

    Rice, E W; Sowers, E G; Johnson, C H; Dunnigan, M E; Strockbine, N A; Edberg, S C

    1992-05-01

    The antigenic relatedness of Escherichia coli O157 and four sorbitol-negative species of the genus Escherichia was examined. Isolates of Escherichia hermannii, E. fergusonii, E. vulneris, and E. blattae were tested in the tube agglutination assay by using polyclonal antisera and in the slide agglutination assay by using latex reagents. Only four isolates (17%) of E. hermannii exhibited serological cross-reactivity. PMID:1583138

  12. Serological cross-reactions between Escherichia coli O157 and other species of the genus Escherichia.

    PubMed Central

    Rice, E W; Sowers, E G; Johnson, C H; Dunnigan, M E; Strockbine, N A; Edberg, S C

    1992-01-01

    The antigenic relatedness of Escherichia coli O157 and four sorbitol-negative species of the genus Escherichia was examined. Isolates of Escherichia hermannii, E. fergusonii, E. vulneris, and E. blattae were tested in the tube agglutination assay by using polyclonal antisera and in the slide agglutination assay by using latex reagents. Only four isolates (17%) of E. hermannii exhibited serological cross-reactivity. PMID:1583138

  13. The Biology of the Escherichia coli Extracellular Matrix

    PubMed Central

    Hufnagel, David A.; DePas, William H.; Chapman, Matthew R.

    2015-01-01

    Chapter Summary Escherichia coli (E. coli) is one of the world’s best-characterized organisms, as it has been extensively studied for over a century. However, most of this work has focused on E. coli grown under laboratory conditions that do not faithfully simulate its natural environments. Therefore, the historical perspectives on E. coli physiology and life cycle are somewhat skewed toward experimental systems that feature E. coli growing logarithmically in a test tube. Typically a commensal bacterium, E. coli resides in the lower intestines of a slew of animals. Outside of the lower intestine, E. coli can adapt and survive in a very different set of environmental conditions. Biofilm formation allows E. coli to survive, and even thrive, in environments that do not support the growth of planktonic populations. E. coli can form biofilms virtually everywhere; in the bladder during a urinary tract infection, on in-dwelling medical devices, and outside of the host on plants and in the soil. The E. coli extracellular matrix, primarily composed of the protein polymer named curli and the polysaccharide cellulose, promotes adherence to organic and inorganic surfaces, and resistance to desiccation, the host immune system and other antimicrobials. The pathways that govern E. coli biofilm formation, cellulose production, and curli biogenesis will be discussed in this book chapter, which concludes with insights into the future of E. coli biofilm research and potential therapies. PMID:26185090

  14. The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli

    PubMed Central

    Balakrishnan, Rohan; Oman, Kenji; Shoji, Shinichiro; Bundschuh, Ralf; Fredrick, Kurt

    2014-01-01

    LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine–Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes. PMID:25378333

  15. Binding studies of antimicrobial peptides to Escherichia coli cells.

    PubMed

    Avitabile, Concetta; D'Andrea, Luca D; Saviano, Michele; Olivieri, Michele; Cimmino, Amelia; Romanelli, Alessandra

    2016-09-01

    Understanding the mechanism of action of antimicrobial peptides is pivotal to the design of new and more active peptides. In the last few years it has become clear that the behavior of antimicrobial peptides on membrane model systems does not always translate to cells; therefore the need to develop methods aimed at capturing details of the interactions of peptides with bacterial cells is compelling. In this work we analyzed binding of two peptides, namely temporin B and TB_KKG6A, to Escherichia coli cells and to Escherichia coli LPS. Temporin B is a natural peptide active against Gram positive bacteria but inactive against Gram negative bacteria, TB_KKG6A is an analogue of temporin B showing activity against both Gram positive and Gram negative bacteria. We found that binding to cells occurs only for the active peptide TB_KKG6A; stoichiometry and affinity constant of this peptide toward Escherichia coli cells were determined. PMID:27450805

  16. The quaternary structure of the ribosome from E. coli. A neutron small-angle scattering study

    NASA Astrophysics Data System (ADS)

    Nowotny, V.; Nowotny, P.; Voß, H.; Nierhaus, K. H.; May, R. P.

    1989-01-01

    Ribosomes synthesize proteins in living cells. The E. coli ribosome is composed of a small (30S) and a large subunit (50S). They consist of different proteins (21 or 34, respectively) and of ribosomal RNAs (16S or 23S and 5S). The inter-protein distances within the ribosomal subunits can be measured from scattering experiments with selectively labeled protein pairs from which the quaternary distribution of the proteins is reconstructed. We have developed the strategy of the “glassy ribosome”: the rRNAs and the proteins are deuterated such that they reach the same scattering density and are “invisible” in a corresponding buffer solution. A preliminary quaternary map of the 50S subunit which is the result of our new method for the extraction of the distances from the scattering data as well as shape parameters of proteins in situ will be presented.

  17. 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

  18. Recurrent Hemolytic and Uremic Syndrome Induced by Escherichia Coli.

    PubMed

    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

    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

  19. Recent advances in adherence and invasion of pathogenic Escherichia coli

    PubMed Central

    Kalita, Anjana; Hu, Jia; Torres, Alfredo G.

    2014-01-01

    Purpose of review Colonization of the host epithelia by pathogenic Escherichia coli is influenced by the ability of the bacteria to interact with host surfaces. Because the initial step of an E. coli infection is to adhere, invade, and persist within host cells, some strategies used by intestinal and extra-intestinal E. coli to infect host cell are presented. Recent findings This review highlights recent progress understanding how extra-intestinal pathogenic E. coli strains express specific adhesins/invasins that allow colonization of the urinary tract or the meninges, while intestinal E. coli strains are able to colonize different regions of the intestinal tract using other specialized adhesins/invasins. Finally, evaluation of, different diets and environmental conditions regulating the colonization of these pathogens is discussed. Summary Discovery of new interactions between pathogenic E. coli and the host epithelial cells unravels the need of more mechanistic studies that can provide new clues in how to combat these infections. PMID:25023740

  20. Virulence attributes of Escherichia coli isolated from dairy heifer feces.

    PubMed

    Cray, W C; Thomas, L A; Schneider, R A; Moon, H W

    1996-12-01

    Escherichia coli isolates from 1,305 (of 6,894) fecal samples collected during the 1991-1992 USDA, Animal and Plant Health Inspection Service, National Health Monitoring System, Diary Heifer Evaluation Project were tested for virulence attributes associated with human enterohaemorrhagic E. coli (EHEC) and the enterotoxin commonly associated with diarrhoea in newborn calves. Single, random isolates from each heifer were hybridized to probes derived from the 60 mDa EHEC plasmid (CVD 419), E. coli attaching and effacing gene (eae), Shiga-like toxin (slt) genes I and II, and E. coli heat-stable enterotoxin a (STaP). Seventy-seven of the 1305 isolates (5.9%) were slt-positive. Most (81.8%) slt-positive E. coli were also CVD 419 and eae-positive. Only 2 of the slt-positive E. coli isolates were STaP-positive. PMID:9008347

  1. Transcription of foreign DNA in Escherichia coli

    PubMed Central

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

    2008-01-01

    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 σ70 (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. PMID:18701636

  2. Genes and proteins of Escherichia coli K-12.

    PubMed

    Riley, M

    1998-01-01

    GenProtEC is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins, representing groups of paralogous genes, with PAM values, percent identity of amino acids, length of alignment and percent aligned. GenProtEC can be accessed at the URL http://www.mbl.edu/html/ecoli.html PMID:9399799

  3. Antibacterial activity and inhibition of protein synthesis in Escherichia coli by antisense DNA analogs.

    PubMed

    Rahman, M A; Summerton, J; Foster, E; Cunningham, K; Stirchak, E; Weller, D; Schaup, H W

    1991-01-01

    Protein synthesis, which takes place within ribosomes, is essential for the survival of any living organism. Ribosomes are composed of both proteins and RNA. Specific interaction between the 3' end CCUCC sequence of prokaryotic 16S rRNA and a partially complementary sequence preceding the initiating codon of mRNA is believed to be a prerequisite for initiation of protein synthesis. Here we report the use of short (three to six nucleotides) synthetic DNA analogs complementary to this sequence to block protein synthesis in vitro and in vivo in Escherichia coli. In the DNA analogs the normal phosphodiester bond in the antisense DNA was replaced by methylcarbamate internucleoside linkages to enhance transport across plasma membranes. Of the analogs tested, those with the sequence AGG and GGA inhibit protein synthesis and colony formation by E. coli strains lacking an outer cell wall. Polyethylene glycol 1000 (PEG 1000) was attached to the 5' end of some of the test methylcarbamate DNAs to enhance solubility. Analogs of AGG and GGAG with PEG 1000 attached inhibited colony formation in normal E. coli. These analogs may be useful food additives to control bacterial spoilage and biomedically as antibiotics. PMID:1821653

  4. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    SciTech Connect

    Svergun, D.I.; Koch, M.H.J.; Pedersen, J.S.; Serdyuk, I.N.

    1994-12-31

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

  5. 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. . Dept. of Genetics); 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.

  6. 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. PMID:25482085

  7. YeeO from Escherichia coli exports flavins

    PubMed Central

    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. PMID:25482085

  8. Characterization of pili associated with Escherichia coli O18ac.

    PubMed Central

    Wevers, P; Picken, R; Schmidt, G; Jann, B; Jann, K; Golecki, J R; Kist, M

    1980-01-01

    A strain of Escherichia coli O18ac isolated from the stool sample of a patient with diarrhea was found to agglutinate human erythrocytes. From the results presented it is suggested that this hemagglutination is mediated by pili. Isolated pilus preparations agglutinated human erythrocytes, whereas pilus-negative mutants did not. The serological and chemical analyses indicate that the pili associated with E. coli O18ac are distinct from other types found with E. coli. Images Fig. 1 Fig. 2 Fig. 3 PMID:6111534

  9. 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

  10. 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

  11. Solution Structure of an Alternate Conformation of Helix 27 from Escherichia Coli 16S rRNA†

    PubMed Central

    Spano, Meredith Newby; Walter, Nils G.

    2011-01-01

    Helix (H)27 of 16S ribosomal (r)RNA from Escherichia coli was dubbed the “switch helix” when mutagenesis suggested that two alternative base pair registers may have distinct functional roles in the bacterial ribosome. Although more recent genetic analyses suggest that H27 conformational switching is not required for translation, previous solution studies demonstrated that the isolated E. coli H27 can dynamically convert between the 885 and 888 conformations. Here, we have solved the NMR solution structure of a locked 888 conformation. NOE and residual dipolar coupling restraints reveal an architecture that markedly differs from that of the 885 conformation found in crystal structures of the bacterial ribosome. In place of the loop E motif that characterizes the 885 conformer and that the 888 conformer cannot adopt, we find evidence for an asymmetrical A-rich internal loop stabilized by stacking interactions among the unpaired A’s. Comparison of the isolated H27 888 solution structure with the 885 crystal structure within the context of the ribosome suggests a difference in overall length of H27 that presents one plausible reason for the absence of H27 conformational switching within the sterically confining ribosome. PMID:21442607

  12. Molecular characterization of diarrheagenic Escherichia coli from Libya.

    PubMed

    Ali, Mostafa Mohamed M; Mohamed, Zienat Kamel; Klena, John D; Ahmed, Salwa Fouad; Moussa, Tarek A A; Ghenghesh, Khalifa Sifaw

    2012-05-01

    Diarrheagenic Escherichia coli (DEC) are important enteric pathogens that cause a wide variety of gastrointestinal diseases, particularly in children. Escherichia coli isolates cultured from 243 diarrheal stool samples obtained from Libyan children and 50 water samples were screened by polymerase chain reaction (PCR) for genes characteristic of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (EIEC). The DEC were detected in 21 (8.6%) children with diarrhea; 10 (4.1%) cases were identified as EAEC, 3 (1.2%) as EPEC, and 8 (3.3%) were ETEC; EHEC, and EIEC were not detected. All DEC were grouped phylogenetically by PCR with the majority (> 70%) identified as phylogenetic groups A and B1. The EAEC isolates were also tested for eight genes associated with virulence using PCR. Multi-virulence (≥ 3 virulence factors) was found in 50% of EAEC isolates. Isolated EAEC possessed different virulence traits and belonged to different phylogenetic groups indicating their heterogeneity. PMID:22556089

  13. Molecular Characterization of Diarrheagenic Escherichia coli from Libya

    PubMed Central

    Ali, Mostafa Mohamed M.; Mohamed, Zienat Kamel; Klena, John D.; Ahmed, Salwa Fouad; Moussa, Tarek A. A.; Ghenghesh, Khalifa Sifaw

    2012-01-01

    Diarrheagenic Escherichia coli (DEC) are important enteric pathogens that cause a wide variety of gastrointestinal diseases, particularly in children. Escherichia coli isolates cultured from 243 diarrheal stool samples obtained from Libyan children and 50 water samples were screened by polymerase chain reaction (PCR) for genes characteristic of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (EIEC). The DEC were detected in 21 (8.6%) children with diarrhea; 10 (4.1%) cases were identified as EAEC, 3 (1.2%) as EPEC, and 8 (3.3%) were ETEC; EHEC, and EIEC were not detected. All DEC were grouped phylogenetically by PCR with the majority (> 70%) identified as phylogenetic groups A and B1. The EAEC isolates were also tested for eight genes associated with virulence using PCR. Multi-virulence (≥ 3 virulence factors) was found in 50% of EAEC isolates. Isolated EAEC possessed different virulence traits and belonged to different phylogenetic groups indicating their heterogeneity. PMID:22556089

  14. Non-O157 Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a leading cause of food-borne illness in the United States; however, recent reports have shown that non-O157 STEC serogroups contribute to more illnesses than O157:H7. Illness caused by non-O157 STEC strains are generally less severe than tho...

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

    PubMed

    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; Fanning, Séamus

    2016-09-01

    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

  16. 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...

  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. 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...

  19. Isolation of an Lc-specific Escherichia coli bacteriophage.

    PubMed Central

    Fralick, J A; Diedrich, D L; Casey-Wood, S

    1990-01-01

    We isolated an OmpF-specific bacteriophage whose host range mutant, SQ108h2, requires the presence of the Lc porin for its attachment and which can be used to screen or select for Lc-defective mutants among Escherichia coli K-12 strains lysogenic for the PA-2 converting phage. Images FIG. 1 PMID:1689719

  20. 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...

  1. Genotypic Characterization of Enterotoxigenic Escherichia coli Strains Causing Traveler's Diarrhea

    PubMed Central

    Rivera, Fulton P.; Medina, Anicia M.; Aldasoro, Edelweiss; Sangil, Anna; Gascon, Joaquim; Ochoa, Theresa J.; Vila, Jordi

    2013-01-01

    This study aims to characterize the presence of virulence factors of enterotoxigenic Escherichia coli (ETEC) causing traveler's diarrhea. Among 52 ETEC isolates, the most common toxin type was STh, and the most frequent colonization factors (CFs) were CS21, CS6, and CS3. On the other hand, the nonclassical virulence factors EAST1 and EatA were frequently present. PMID:23224092

  2. Identification of the Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase Gene

    PubMed Central

    Mehl, Ryan A.; Kinsland, Cynthia; Begley, Tadhg P.

    2000-01-01

    The gene (ybeN) coding for nicotinate mononucleotide adenylyltransferase, an NAD(P) biosynthetic enzyme, has been identified and overexpressed in Escherichia coli. This enzyme catalyzes the reversible adenylation of nicotinate mononucleotide and shows product inhibition. The rate of adenylation of nicotinate mononucleotide is at least 20 times faster than the rate of adenylation of nicotinamide mononucleotide. PMID:10894752

  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 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3255...

  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 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3255...

  5. 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 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3255...

  6. 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 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3255...

  7. 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 Section 866.3255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3255...

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

    PubMed

    Coura, Fernanda Morcatti; 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

  9. 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

  10. Effects of low concentrations of antibiotics on Escherichia coli adhesion.

    PubMed Central

    Vosbeck, K; Mett, H; Huber, U; Bohn, J; Petignat, M

    1982-01-01

    We have previously shown that subinhibitory concentrations of antibiotics may influence the adhesion of Escherichia coli SS142 to human epithelioid tissue culture cells. This report shows that these effects are not limited to E. coli SS142 or to our tissue culture system. Most of the 10 E. coli strains studied showed decreased adhesion to Intestine 407 tissue culture cells after growth in 25% of the minimum inhibitory concentration of streptomycin, tetracycline, trimethoprimsulfametrole, chloramphenicol, and clindamycin. Nalidixic acid at 25% of the minimum inhibitory concentration caused an increase of adhesion. The hemagglutinating activity of the five hemagglutinating strains and the adhesiveness of E. coli SS142 to human buccal cells were similarly affected by low concentrations of the above-mentioned antibiotics. We conclude that E. coli adhesion to human epithelioid tissue culture cells is a valid model of bacterial adhesion because of its high accuracy and reproducibility. PMID:7051972

  11. Feedback regulation of the spc operon in Escherichia coli: translational coupling and mRNA processing.

    PubMed Central

    Mattheakis, L C; Nomura, M

    1988-01-01

    The spc operon of Escherichia coli encodes 10 ribosomal proteins in the order L14, L24, L5, S14, S8, L6, L18, S5, L30, and L15. This operon is feedback regulated by S8, which binds near the translation start site of L5 and inhibits translation of L5 directly and that of the distal genes indirectly. We constructed plasmids carrying a major portion of the spc operon genes under lac transcriptional control. The plasmids carried a point mutation in the S8 target site which abolished regulation and resulted in overproduction of plasmid-encoded ribosomal proteins upon induction. We showed that alteration of the AUG start codon of L5 to UAG decreased the synthesis rates of plasmid-encoded distal proteins, as well as L5, by approximately 20-fold, with a much smaller (if any) effect on mRNA synthesis rates, indicating coupling of the distal cistrons' translation with the translation of L5. This conclusion was also supported by experiments in which S8 was overproduced in trans. In this case, there was a threefold reduction in the synthesis rates of chromosome-encoded L5 and the distal spc operon proteins, but no decrease in the mRNA synthesis rate. These observations also suggest that transcription from ribosomal protein promoters may be special, perhaps able to overcome transcription termination signals. We also analyzed the state of ribosomal protein mRNA after overproduction of S8 in these experiments and found that repression of ribosomal protein synthesis was accompanied by stimulation of processing (and degradation) of spc operon mRNA. The possible role of mRNA degradation in tightening the regulation is discussed. Images PMID:3049533

  12. 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

  13. An Escherichia coli Mutant That Makes Exceptionally Long Cells

    PubMed Central

    Newman, Elaine B.

    2015-01-01

    ABSTRACT Although Escherichia coli is a very small (1- to 2-μm) rod-shaped cell, here we describe an E. coli mutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. These extremely elongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division. IMPORTANCE Escherichia coli is usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usual E. coli cell. E. coli filaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects of E. coli physiology that are difficult to investigate with small cells. PMID:25691528

  14. The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli.

    PubMed Central

    Lawrence, J G; Roth, J R

    1995-01-01

    The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria. PMID:7592411

  15. Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

    DOE R&D Accomplishments Database

    Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

    1982-06-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

  16. 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

  17. Functional characterization of alternate optimal solutions of Escherichia coli's transcriptional and translational machinery.

    PubMed

    Thiele, Ines; Fleming, Ronan M T; Bordbar, Aarash; Schellenberger, Jan; Palsson, Bernhard Ø

    2010-05-19

    The constraint-based reconstruction and analysis approach has recently been extended to describe Escherichia coli's transcriptional and translational machinery. Here, we introduce the concept of reaction coupling to represent the dependency between protein synthesis and utilization. These coupling constraints lead to a significant contraction of the feasible set of steady-state fluxes. The subset of alternate optimal solutions (AOS) consistent with maximal ribosome production was calculated. The majority of transcriptional and translational reactions were active for all of these AOS, showing that the network has a low degree of redundancy. Furthermore, all calculated AOS contained the qualitative expression of at least 92% of the known essential genes. Principal component analysis of AOS demonstrated that energy currencies (ATP, GTP, and phosphate) dominate the network's capability to produce ribosomes. Additionally, we identified regulatory control points of the network, which include the transcription reactions of sigma70 (RpoD) as well as that of a degradosome component (Rne) and of tRNA charging (ValS). These reactions contribute significant variance among AOS. These results show that constraint-based modeling can be applied to gain insight into the systemic properties of E. coli's transcriptional and translational machinery. PMID:20483314

  18. Endogenous occurrence of protein S-guanylation in Escherichia coli: Target identification and genetic regulation.

    PubMed

    Tsutsuki, Hiroyasu; Jung, Minkyung; Zhang, Tianli; Ono, Katsuhiko; Ida, Tomoaki; Kunieda, Kohei; Ihara, Hideshi; Akaike, Takaaki; Sawa, Tomohiro

    2016-09-01

    8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E. coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E. coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E. coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E. coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5'-triphosphate. More importantly, E. coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E. coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections. PMID:27473654

  19. The different ecological niches of enterotoxigenic Escherichia coli.

    PubMed

    Gonzales-Siles, Lucia; Sjöling, Åsa

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) is a water and food-borne pathogen that infects the small intestine of the human gut and causes diarrhoea. Enterotoxigenic E. coli adheres to the epithelium by means of colonization factors and secretes two enterotoxins, the heat labile toxin and/or the heat stable toxin that both deregulate ion channels and cause secretory diarrhoea. Enterotoxigenic E. coli as all E. coli, is a versatile organism able to survive and grow in different environments. During transmission and infection, ETEC is exposed to various environmental cues that have an impact on survivability and virulence. The ability to cope with exposure to different stressful habitats is probably shaping the pool of virulent ETEC strains that cause both endemic and epidemic infections. This review will focus on the ecology of ETEC in its different habitats and interactions with other organisms as well as abiotic factors. PMID:26522129

  20. Enteropathogenic Escherichia coli in raw and cooked food.

    PubMed

    Norazah, A; Rahizan, I; Zainuldin, T; Rohani, M Y; Kamel, A G

    1998-03-01

    A total of 402 Escherichia coli isolates were obtained from a variety of food samples and screened for enteropathogenic E. coli (EPEC). Screening was carried out using 15 specific monovalent antisera from Murex Diagnostic Limited. A total of 19 E. coli isolates were serotyped as EPEC. The EPEC strains were shown to belong to 8 serotypes. Eight out of 19 EPEC strains belonged to serotype 018C:K77 (B21). Seventeen out of 19 of the EPEC strains were isolated from cooked food. The presence of E. coli in cooked food is an indicator of fecal contamination and a sign of unhygienic food handling. The presence of EPEC in food could be a potential source of food-borne outbreak. Hygiene training for every food-handler is a necessity. PMID:9740276

  1. Requirements for translation re-initiation in Escherichia coli: roles of initiator tRNA and initiation factors IF2 and IF3

    PubMed Central

    Yoo, Jae-Ho; RajBhandary, Uttam L

    2008-01-01

    Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV–geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA – formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site – are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage. PMID:18221266

  2. 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

  3. Enteropathogenic Escherichia coli Prevalence in Laboratory Rabbits

    PubMed Central

    Swennes, Alton G.; Buckley, Ellen M.; Madden, Carolyn M.; Byrd, Charles P.; Donocoff, Rachel S.; Rodriguez, Loretta; Parry, Nicola M. A.; Fox, James G.

    2013-01-01

    Rabbit-origin enteropathogenic E. coli (EPEC) causes substantial diarrhea-associated morbidity and has zoonotic potential. A culture-based survey was undertaken to ascertain its prevalence. EPEC was isolated from 6/141 (4.3%) commercially-acquired laboratory rabbits. Three of these did not have diarrhea or EPEC-typical intestinal lesions; they instead had background plasmacytic intestinal inflammation. Asymptomatically infected rabbits may function as EPEC reservoirs. PMID:23391439

  4. Resistance and virulence factors of Escherichia coli isolated from chicken.

    PubMed

    Pavlickova, Silvie; Dolezalova, Magda; Holko, Ivan

    2015-01-01

    Chicken meat has become an important part of the human diet and besides contamination by pathogenic Escherichia coli there is a risk of antibiotic resistance spreading via the food chain. The purpose of this study was to examine the prevalence of resistance against eight antibiotics and the presence of 14 virulence factors among 75 Escherichia coli strains isolated from chicken meat in the Czech Republic after classification into phylogenetic groups by the multiplex PCR method. More than half of strains belonged to A phylogroup, next frequently represented was B1 phylogroup, which suggests the commensal strains. The other strains were classified into phylogroups B2 and D, which had more virulence factors. Almost half of all E. coli strains were resistant to at least one of eight-tested antibiotics. A multidrug resistance was observed in 13% of strains. The most prevalent virulence genes were iucD, iss and tsh. None of genes encoding toxins was detected. Most of E. coli strains isolated from chicken meat can be considered as nonpathogenic on the basis of analysis of virulence factors, antibiotic resistance and phylogroups assignment. It can provide a useful tool for prediction of a potential risk from food contaminated by E. coli. PMID:25844863

  5. Copper, zinc superoxide dismutase in Escherichia coli: periplasmic localization.

    PubMed

    Benov, L; Chang, L Y; Day, B; Fridovich, I

    1995-06-01

    Cu,ZnSOD purified from Escherichia coli has been used to raise antibodies in rabbits. The resultant antiserum was found to recognize a single band on Western blots of SDS-polyacrylamide gel electropherograms, and that single band coincided with the position of the Cu,ZnSOD. Ultrathin sections of fixed E. coli were treated with the antibody followed by protein A bearing 10-nm gold particles. Electron microscopy revealed that Cu,ZnSOD was largely localized in the periplasm in polar bays. PMID:7786035

  6. 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. PMID:26310235

  7. Genes and proteins of Escherichia coli (GenProtEc).

    PubMed

    Riley, M; Space, D B

    1996-01-01

    GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned. The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe. The program runs under MS-DOS on IMB-compatible machines. GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html. PMID:8594596

  8. Reduction of methionine sulfoxide to methionine by Escherichia coli.

    PubMed Central

    Ejiri, S I; Weissbach, H; Brot, N

    1979-01-01

    L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine. This was verified by showing that a cell-free extract of E. coli catalyzes the conversion of methionine sulfoxide to methionine. This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound. The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml. PMID:37234

  9. Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli.

    PubMed Central

    Cronan, J E; Reed, R; Taylor, F R; Jackson, M B

    1979-01-01

    The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis. PMID:374358

  10. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  11. 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. PMID:24246520

  12. Dexamethazone protects against Escherichia coli induced sickness behavior in rats.

    PubMed

    Hanaa-Mansour, A; Hassan, Wedad A; Georgy, Gehan S

    2016-01-01

    Systemic bacterial infection results in systemic inflammatory response syndrome due to the release of lipopolysaccharide (LPS) in blood that can lead to multiple organ failure, shock, and potentially death. Other impact, LPS exposure produces robust increase in anxiety-like behavior, suppression of locomotor, exploratory activity, and reduced social behavior. The therapeutic use of glucocorticoids in septic shock remains one of the first-aid approaches for their anti-inflammatory properties. The aim of this study was to evaluate the possible protective effect of dexamethazone (DEX), the most commonly used corticosteroid, against Escherichia coli (E. coli) immunohistochemical changes and neurobehavioral dysfunction. To this end, male Sprague-Dawley rats were divided into four groups; (1) Control group (2) E. coli infected group, where animals received 0.2 ml of 24 h growth of E. coli suspension in nutrient broth containing approximately 1.8×10(8) cfu/ml i.p for once, 48 h before sacrificing (3) DEX (20 mg/kg, i.p, 3 days) treated group (4) DEX and E. coli treated group. The results revealed that DEX significantly protected animals against most E. coli-induced behavioral deficits, reduced signs of cognitive impairment. DEX also reduced the LPS-evoked rise in C-reactive protein (CRP), Interferon gamma (IFγ), as well as, expression of Caspase-3. In conclusion, DEX provides neuroprotection against E. coli-associated neurobehavioral and immunological changes via its anti-inflammatory and immunomodulatory effects. PMID:26541583

  13. Pathotyping blaCTX-M Escherichia coli from Nigeria

    PubMed Central

    Olowe, Olugbenga Adekunle; Choudhary, Suman; Schierack, Peter; Wieler, Lothar H.; Olayemi, Albert B.; Anjum, Muna

    2013-01-01

    Background: Escherichia coli have become the enterobacteriaceae species most affected by extended-spectrum β-lactamases (ESBLs) in view of the emergence of CTX-M-type ESBLs. These CTX-M-positive E. coli have been reported in numerous regions worldwide. Virulence determinants of already reported CTX-M-positive E. coli were investigated. Methodology: To gain insights into the mechanism underlying this phenomenon, we assessed serogroup, susceptibility pattern and diversity of virulence profiles within a collection of nine blaCTX-M-positive E. coli strains and their virulent determinant using miniaturized DNA microarray techniques. The nine ESBL-positive E. coli isolates were from eight male and one female patient(s) selected for study based on previous work. Virulence potential was inferred by detection of 63 virulence factor (VF) genes. Results: Four (44.4%) of the 9 E. coli isolates exhibited the same set of core characteristics: serotype O8:Hnt, while all were positive for OXA-1, ciprofloxacin resistance. Five of the isolates exhibited highly similar (91% to 100%) VF profiles. Conclusion: The findings describe a broadly disseminated, blaCTX-M-positive and virulent E. coli serogroup with highly homogeneous virulence genotypes, suggesting recent emergence in this zone. Understanding how this clone has emerged and successfully disseminated within the hospital and community, including across national boundaries, should be a public health priority. PMID:24265928

  14. Advances in molecular serotyping and subtyping of Escherichia coli

    DOE PAGESBeta

    Fratamico, Pina M.; DebRoy, Chitrita; Liu, Yanhong; Needleman, David S.; Baranzoni, Gian Marco; Feng, Peter

    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

  15. An Archaea 5S rRNA analog is stably expressed in Escherichia coli

    NASA Technical Reports Server (NTRS)

    Yang, Y.; Fox, G. E.

    1996-01-01

    Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

  16. 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

  17. Two-dimensional polyacylamide gel electrophoresis of envelope proteins of Escherichia coli.

    PubMed

    Johnson, W C; Silhavy, T J; Boos, W

    1975-03-01

    A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight. PMID:803821

  18. Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

    PubMed Central

    Rosano, C L; Bunce, S C; Hurwitz, C

    1983-01-01

    At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes. PMID:6336736

  19. Purification of DNA for bacterial productivity estimates. [Escherichia coli

    SciTech Connect

    Burnison, B.K.; Nuttley, D.J. )

    1990-02-01

    (methyl-{sup 3}H)thymidine-labeled DNA from natural populations of aquatic bacteria was completely separated from RNA and protein by hydroxylapatite chromatography. The procedure was validated by monitoring increases in Escherichia coli cell count, A{sup 550}, DNA concentration, and thymidine incorporation into DNA isolated by the proposed technique. The procedure can be used in the field and does not rely on the use of acid-base hydrolysis or volatile organic solvents.

  20. Genetic Analysis of the Maltose A Region in Escherichia coli

    PubMed Central

    Hatfield, Dolph; Hofnung, Maurice; Schwartz, Maxime

    1969-01-01

    The genetic map of the maltose A locus of Escherichia coli contains at least three closely linked genes, malT, malP, and malQ. The order of these genes is established by deletion mapping. MalP and malQ, the presumed structural genes for maltodextrin phosphorylase and amylomaltase, belong to the same operon. MalT may be a regulator gene involved in the positive control of this operon. PMID:4891257

  1. ¹³C-metabolic flux analysis for Escherichia coli.

    PubMed

    Matsuoka, Yu; Shimizu, Kazuyuki

    2014-01-01

    (13)C-Metabolic flux analysis ((13)C-MFA) is used here to study the effects of the knockout of such genes as pgi, zwf, gnd, ppc, pck, pyk, and lpdA on the metabolic changes in Escherichia coli cultivated under aerobic condition. The metabolic regulation mechanisms were clarified by integrating such information as fermentation data, gene expression, enzyme activities, and metabolite concentrations as well the result of (13)C-MFA. PMID:25178796

  2. Electric field induced bacterial flocculation of enteroaggregative Escherichia coli 042

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

    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 heterogenous 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.

  3. Occurrence of Escherichia coli in Wild Cottontail Rabbits.

    PubMed

    Kozlowski, R; Glantz, P J; Anthony, R G

    1977-03-01

    Free-ranging cottontail rabbits (Sylvilagus floridanus) from two areas in central Pennsylvania were sampled over a 4-year period. Large numbers of coliforms were isolated from the intestinal tracts of these animals; in 136 of the 141 rabbits sampled, Escherichia coli was found to be a major component of the alimentary flora. Four serogroups (O7, O77, O73, and O103) were predominant among the isolates and were considered resistant coliflora of this species of cottontail rabbit. PMID:16345208

  4. Role of threonine dehydrogenase in Escherichia coli threonine degradation.

    PubMed Central

    Potter, R; Kapoor, V; Newman, E B

    1977-01-01

    Threonine was used as nitrogen source by Escherichia coli K-12 through a pathway beginning with the enzyme threonine dehydrogenase. The 2-amino-3-ketobutyrate formed was converted to glycine, and the glycine was converted to serine, which acted as the actual nitrogen donor. The enzyme formed under anaerobic conditions and known as threonine deaminase (biodegradative) is less widespread than threonine dehydrogenase and may be involved in energy metabolism rather than in threonine degradation per se. PMID:334738

  5. 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.

  6. Anatomy of Escherichia coli σ70 promoters

    PubMed Central

    Shultzaberger, Ryan K.; Chen, Zehua; Lewis, Karen A.; Schneider, Thomas D.

    2007-01-01

    Information theory was used to build a promoter model that accounts for the −10, the −35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base −7, rather than −11, of the −10 may be flipping to initiate transcription. We found that the sequence conservation of σ70 binding sites is 6.5 ± 0.1 bits. Some promoters lack a −35 region, but have a 6.7 ± 0.2 bit extended −10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended −10 binding and the −35 suggest that the flexible bipartite σ factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5′ mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the −35, so bending allows distal activators to reach the polymerase. PMID:17189297

  7. Comparison of three types of biochar for removal of Escherichia coli from agricultural runoff

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) is an infectious type of bacteria that infects over 5,000 people per year in the United States, sometimes leading to death. Since cattle can produce more than 104 Escherichia coli (E. coli) per gram of feces, and biochar is a material with physical prop...

  8. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed.

    PubMed

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  9. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  10. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    PubMed Central

    Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. PMID:25438014

  11. Analysis of plasmids cloned from a virulent avian Escherichia coli and transformed into Escherichia coli DH5 alpha.

    PubMed

    Wooley, R E; Gibbs, P S; Dickerson, H W; Brown, J; Nolan, L K

    1996-01-01

    Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha. PMID:8883780

  12. Escherichia coli heme oxygenase modulates host innate immune responses

    PubMed Central

    Maharshak, Nitsan; Ryu, Hyungjin Sally; Fan, Ting-Jia; Onyiah, Joseph C.; Schulz, Stephanie; Otterbein, Sherrie L.; Wong, Ron; Hansen, Jonathan; Otterbein, Leo E; Carroll, Ian; Plevy, Scott E.

    2015-01-01

    Induction of mammalian heme oxygenase-1 and exposure of animals to carbon monoxide ameliorates experimental colitis. When enteric bacteria, including Escherichia coli, are exposed to low iron conditions, they express an heme oxygenase-like enzyme, chuS, and metabolize heme into iron, biliverdin and carbon monoxide. Given the abundance of enteric bacteria residing in the intestinal lumen, we hypothesized that commensal intestinal bacteria may be a significant source of carbon monoxide, with the consequence that enteric bacteria expressing chuS and other heme oxygenase -like molecules suppress inflammatory immune responses through release of carbon monoxide. Carbon monoxide exposed mice have altered enteric bacterial composition and increased E. coli 16S and chuS DNA by real-time PCR. Moreover, severity of experimental colitis correlates with increased E. coli chuS expression in IL-10 deficient mice. To explore functional roles, E. coli were genetically modified to overexpress chuS or the chuS gene was deleted. Co-culture of chuS-overexpressing E. coli with bone marrow derived macrophages results in decreased IL-12 p40 and increased IL-10 secretion compared to wild-type or chuS-deficient E. coli. Mice infected with chuS-overexpressing E. coli have increased levels of hepatic carbon monoxide and decreased serum IL-12 p40 compared to mice infected with chuS-deficient E. coli. Thus, carbon monoxide alters the composition of the commensal intestinal microbiota and expands E. coli populations harboring the chuS gene. These bacteria are capable of attenuating innate immune responses through expression of chuS. Bacterial heme oxygenase -like molecules and bacterial-derived carbon monoxide may represent novel targets for therapeutic intervention in inflammatory conditions. PMID:26146866

  13. Expression of the Serratia marcescens lipoproteins gene in Escherichia coli.

    PubMed Central

    Lee, N; Nakamura, K; Inouye, M

    1981-01-01

    The lipoprotein gene (lpp) of Serratia marcescens was cloned in a lambda phage vector (K. Nakamura and M. Inouye, Proc. Natl. Acad. Sci. U.S.A. 77: 1369-1373, 1980). This lpp gene was recloned in plasmid vectors pBR322 and pSC101. When a lipoprotein-deficient (lpp) mutant of Escherichia coli was transformed with pBR322 carrying the S. marcescens lpp gene, cells became nonleaky for ribonuclease, resistant to ethylenediaminetetraacetic acid, and sensitive to globomycin. The lipoprotein was found exclusively in the outer membrane fraction. These results indicate that the S. marcescens lipoprotein was normally secreted across the cytoplasmic membrane, modified, and assembled in the E. coli outer membrane. The amount of the free-form lipoprotein produced in this system was three times higher than that produced in lpp + C. coli cells, whereas there was no difference in the amount of the bound-form lipoprotein. On the other hand, lpp E. coli cells which harbored pSC101 carrying the S. marcescens lpp gene produced only one-third of the free-form lipoprotein produced in lpp E. coli cells which harbored pSC101 carrying the E. coli lpp gene. One of the major factors causing this difference in efficiency of gene expression between the lpp genes of S. marcescens and E. coli appears to be a deletion mutation at the transcription termination region found in the cloned S. marcescens lpp gene. The functional half-life of the S. marcescens lpp messenger ribonucleic acid in E. coli was found to be found half that of the E. coli lpp messenger ribonucleic acid. Images PMID:7016834

  14. Preparation of Soluble Proteins from Escherichia coli.

    PubMed

    Wingfield, Paul T

    2014-01-01

    Purification of human IL-1β is used in this unit as an example of the preparation of a soluble protein 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. In addition, the purification procedure serves as an example of how to use classical protein purifications methods, which may also be used in conjunction with the affinity-based methods now more commonly used. PMID:25367009

  15. 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

  16. 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. PMID:24694052

  17. Bacteriophage cocktail significantly reduces Escherichia coli O157

    PubMed Central

    Carter, Chandi D.; Parks, Adam; Abuladze, Tamar; Li, Manrong; Woolston, Joelle; Magnone, Joshua; Senecal, Andre; Kropinski, Andrew M.; Sulakvelidze, Alexander

    2012-01-01

    Foods contaminated with Escherichia coli O157:H7 cause more than 63,000 foodborne illnesses in the United States every year, resulting in a significant economic impact on medical costs and product liabilities. Efforts to reduce contamination with E. coli O157:H7 have largely focused on washing, application of various antibacterial chemicals, and gamma-irradiation, each of which has practical and environmental drawbacks. A relatively recent, environmentally-friendly approach proposed for eliminating or significantly reducing E. coli O157:H7 contamination of foods is the use of lytic bacteriophages as biocontrol agents. We found that EcoShield™, a commercially available preparation composed of three lytic bacteriophages specific for E. coli O157:H7, significantly (p < 0.05) reduced the levels of the bacterium in experimentally contaminated beef by ≥ 94% and in lettuce by 87% after a five minute contact time. The reduced levels of bacteria were maintained for at least one week at refrigerated temperatures. However, the one-time application of EcoShield™ did not protect the foods from recontamination with E. coli O157:H7. Our results demonstrate that EcoShield™ is effective in significantly reducing contamination of beef and lettuce with E. coli O157:H7, but does not protect against potential later contamination due to, for example, unsanitary handling of the foods post processing. PMID:23275869

  18. 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

  19. 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

  20. Transformation of Escherichia coli and protein expression using lipoplex mimicry.

    PubMed

    Yun, Chul-Ho; Bae, Chun-Sik; Ahn, Taeho

    2016-11-01

    We investigated a "one-step" method for transformation of and protein expression in Escherichia coli (E. coli) using a complex of n-stearylamine, a cationic lipid, and plasmid DNA, which mimics lipoplex-based approaches. When E. coli cells were treated with the cationic lipid-plasmid complex, the transformation efficiencies were in the range of approximately 2-3 × 10(6) colony-forming units. Further increase in the efficiency was obtained by co-treatment with calcium chloride (or rubidium chloride) and the complexes. Moreover, after DNA transfer, E. coli cells successfully expressed plasmid-encoded proteins such as cytochrome P450s and glutathione-S-transferase without overnight incubation of the cells to form colonies, an indispensable step in other bacterial transformation methods. In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step. PMID:27416742

  1. EcoCyc: A comprehensive view of Escherichia coli biology

    PubMed Central

    Keseler, Ingrid M.; Bonavides-Martínez, César; Collado-Vides, Julio; Gama-Castro, Socorro; Gunsalus, Robert P.; Johnson, D. Aaron; Krummenacker, Markus; Nolan, Laura M.; Paley, Suzanne; Paulsen, Ian T.; Peralta-Gil, Martin; Santos-Zavaleta, Alberto; Shearer, Alexander Glennon; Karp, Peter D.

    2009-01-01

    EcoCyc (http://EcoCyc.org) provides a comprehensive encyclopedia of Escherichia coli biology. EcoCyc integrates information about the genome, genes and gene products; the metabolic network; and the regulatory network of E. coli. Recent EcoCyc developments include a new initiative to represent and curate all types of E. coli regulatory processes such as attenuation and regulation by small RNAs. EcoCyc has started to curate Gene Ontology (GO) terms for E. coli and has made a dataset of E. coli GO terms available through the GO Web site. The curation and visualization of electron transfer processes has been significantly improved. Other software and Web site enhancements include the addition of tracks to the EcoCyc genome browser, in particular a type of track designed for the display of ChIP-chip datasets, and the development of a comparative genome browser. A new Genome Omics Viewer enables users to paint omics datasets onto the full E. coli genome for analysis. A new advanced query page guides users in interactively constructing complex database queries against EcoCyc. A Macintosh version of EcoCyc is now available. A series of Webinars is available to instruct users in the use of EcoCyc. PMID:18974181

  2. Virulence of Shigella flexneri Hybrids Expressing Escherichia coli Somatic Antigens

    PubMed Central

    Gemski, P.; Sheahan, D. G.; Washington, O.; Formal, S. B.

    1972-01-01

    The genes controlling either Escherichia coli somatic antigen 8 or 25 were conjugally transferred to virulent Shigella flexneri 2a recipients to determine whether the aquisition of these antigens would affect the virulence of the resulting hybrid. A high proportion of such hybrids were found to be rough and hence were avirulent. Some smooth S. flexneri hybrids which replaced their native group antigens with E. coli factor 25 were still virulent in the animal models employed. All S. flexneri O-8 hybrids were uniformly avirulent. Our finding, that S. flexneri hybrids with the chemically divergent E. coli O-8 repeat unit are avirulent whereas some hybrids with the chemically related O-25 repeat unit retain virulence, suggests that the chemical composition and structure of the O side chain of somatic antigens may represent one determining factor for bacterial penetration of mucosal epithelial cells, the primary step in the pathogenesis of bacillary dysentery. Images PMID:4569915

  3. 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

  4. Reconstruction of a chromatic response system in Escherichia coli.

    PubMed

    Sugie, Yoshimi; Hori, Mayuko; Oka, Shunsuke; Ohtsuka, Hokuto; Aiba, Hirofumi

    2016-07-14

    Two-component signal transduction systems (TCS) are involved in widespread cellular responses to diverse signals from bacteria to plants. Cyanobacteria have evolved photoperception systems for efficient photosynthesis, and some histidine kinases are known to function as photosensors. In this study, we attempt to reconstruct the photoperception system in Escherichia coli to make an easily controllable ON/OFF switch for gene expressions. For this purpose, a CcaS-CcaR two-component system from Nostoc punctiforme was expressed with phycocyanobilin (PCB) producing enzymes in E. coli which carries a G-box-controlled reporter gene. We succeeded to endow E. coli with a gene activation switch that is regulated in a light-color dependent manner. The possibility of such a switch for the development of synthetic biology is pointed out. PMID:27246537

  5. 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

  6. Structural Insights Into Ribosome Recycling Factor Interactions with the 70S Ribosome

    PubMed Central

    Pai, Raj D.; Zhang, Wen; Schuwirth, Barbara S.; Hirokawa, Go; Kaji, Hideko; Kaji, Akira; Cate, Jamie H.D.

    2009-01-01

    SUMMARY At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with Elongation Factor G (EF-G) to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center (PTC). Upon binding of either E. coli or T. thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix H69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits, termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of H69 involves an ordered to disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between Domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling. PMID:18234219

  7. 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...

  8. Global dissemination of a multidrug resistant Escherichia coli clone

    PubMed Central

    Petty, Nicola K.; Ben Zakour, Nouri L.; Stanton-Cook, Mitchell; Skippington, Elizabeth; Totsika, Makrina; Forde, Brian M.; Phan, Minh-Duy; Gomes Moriel, Danilo; Peters, Kate M.; Davies, Mark; Rogers, Benjamin A.; Dougan, Gordon; Rodriguez-Baño, Jesús; Pascual, Alvaro; Pitout, Johann D. D.; Upton, Mathew; Paterson, David L.; Walsh, Timothy R.; Schembri, Mark A.; Beatson, Scott A.

    2014-01-01

    Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000–2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL–resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen. PMID:24706808

  9. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates.

    PubMed

    Johnson, Timothy J; Wannemuehler, Yvonne M; Johnson, Sara J; Logue, Catherine M; White, David G; Doetkott, Curt; Nolan, Lisa K

    2007-03-01

    Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae. Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli. IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC, cbi, and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations. PMID:17277222

  10. 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. PMID:15261962

  11. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications

    PubMed Central

    Gänzle, Michael; Liu, Yang

    2015-01-01

    High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods. PMID:26157424

  12. 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. PMID:22221349

  13. The 9S RNA precursor of Escherichia coli 5S RNA has three structural domains: implications for processing.

    PubMed Central

    Christiansen, J

    1988-01-01

    The secondary structure of the 9S RNA precursor to ribosomal 5S RNA in Escherichia coli has been determined using chemical reagents and ribonucleases in combination with a reverse transcription procedure. The 9S RNA precursor was generated in vitro by T7 RNA polymerase, and the rrnB operon terminator, T1, was able to terminate the in vitro transcript. The secondary structure model exhibits three structural domains corresponding to a 5' region, a mature region and a terminator region. The mature domain is structurally identical to 5S RNA, and the ribosomal proteins L18 and L25 are able to bind to the precursor. The processing endoribonuclease RNase E cleaves between the structural domains. Moreover, an intramolecular refolding of the nascent transcript must take place if the current view of RNase III processing stems is correct. Images PMID:3045757

  14. 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. PMID:27081680

  15. 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

  16. Comparative Genomic Indexing Reveals the Phylogenomics of Escherichia coli Pathogens

    PubMed Central

    Anjum, Muna F.; Lucchini, Sacha; Thompson, Arthur; Hinton, Jay C. D.; Woodward, Martin J.

    2003-01-01

    The Escherichia coli O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing E. coli O26 strains represent the same clonal lineage and that VT-expressing E. coli O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the E. coli species and identified regions of variation from the E. coli K-12 chromosomal backbone. PMID:12874348

  17. 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

  18. Role of peripheral pooling in porcine Escherichia coli sepsis

    SciTech Connect

    Teule, G.J.; von Lingen, A.; Verwey von Vught, M.A.; Kester, A.D.; Mackaay, R.C.; Bezemer, P.D.; Heidenal, G.A.; Thijs, L.G.

    1984-01-01

    In anesthesized pigs the effects of E. coli (2 X 10(8)/kg) on hemodynamics and red cell distribution were studied. After injection of 99m-Tc red cells (15 mCi), regional radioactivity was followed during 3 hours. Gated bloodpool studies were performed to measure end-diastolic volumes (EDV). Escherichia coli E. coli was infused in 14 pigs, while 7 animals served as controls. E. coli resulted in an early increase in pulmonary arterial pressure. Systemic arterial pressure decreased gradually, while cardiac output did not change significantly. The gated studies revealed that especially left ventricular end-diastolic volume (LVEDV) declined, to 50% of the basal value. Regional radioactivity did not change over lungs, liver and abdomen. Splenic activity declined markedly. Over the hindlimb a significant increase (29 +/- 8%) was observed. It is concluded that E. coli infusion in pigs induces a hemodynamic pattern similar to human sepsis. The decrease in LVEDV is probably related to peripheral pooling and a change in right ventricle (RV) performance.

  19. 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

  20. 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

  1. Attachment of Escherichia coli and enterococci to particles in runoff.

    PubMed

    Soupir, Michelle L; Mostaghimi, Saied; Dillaha, Theo

    2010-01-01

    Association of Escherichia coli and enterococci with particulates present in runoff from erodible soils has important implications for modeling the fate and transport of bacteria from agricultural sources and in the selection of management practices to reduce bacterial movement to surface waters. Three soils with different textures were collected from the Ap horizon (silty loam, silty clay loam, and loamy fine sand), placed in portable box plots, treated with standard cowpats, and placed under a rainfall simulator. Rainfall was applied to the plots until saturation-excess flow occurred for 30 min, and samples were collected 10, 20, and 30 min after initiation of the runoff event. The attachment of E. coli and enterococci to particles present in runoff was determined by a screen filtration and centrifugation procedure. Percentage of E. coli and enterococci attached to particulates in runoff ranged from 28 to 49%, with few statistically significant differences in attachment among the three soils. Similar partitioning release patterns were observed between E. coli and enterococci from the silty loam (r = 0.57) and silty clay loam soils (r = 0.60). At least 60% of all attached E. coli and enterococci were associated particles within an 8- to 62-microm particle size category. The results indicate that the majority of fecal bacteria attach to and are transported with manure colloids in sediment-laden flow regardless of the soil texture. PMID:20400597

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

    PubMed Central

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

    2015-01-01

    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. PMID:26410828

  3. Escherichia coli O157 and other Shiga toxin producting E. coli: detection by immunomagnetic particle-based assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7 and non-O157 STEC cause hemorrhagic colitis and hemolytic uremic syndrome and are important food-borne pathogens that can contaminate various types of food. The USDA Food Safety and Inspection Service declared E. coli O157:H7 a...

  4. 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. PMID:26410828

  5. 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. PMID:27499290

  6. Widespread antibiotic resistance of diarrheagenic Escherichia coli and Shigella species

    PubMed Central

    Sadeghabadi, Azam Fatahi; Ajami, Ali; Fadaei, Reza; Zandieh, Masoud; Heidari, Elham; Sadeghi, Mahmoud; Ataei, Behrooz; Hoseini, Shervin Ghaffari

    2014-01-01

    Background: Antibiotic resistance of enteric pathogens particularly Shigella species, is a critical world-wide problem and monitoring their resistant pattern is essential, because the choice of antibiotics is absolutely dependent on regional antibiotic susceptibility patterns. During summer 2013, an unusual increase in number of diarrheal diseases was noticed in Isfahan, a central province of Iran. Therefore, the antibiotic resistance of diarrheagenic Escherichia coli and Shigella species isolated were evaluated. Materials and Methods: According to the guideline on National Surveillance System for Foodborn Diseases, random samples from patients with acute diarrhea were examined in local laboratories of health centers and samples suspicious of Shigella spp. were further assessed in referral laboratory. Isolated pathogens were identified by standard biochemical and serologic tests and antibiotic susceptibility testing was carried out by disc diffusion method. Results: A total of 1086 specimens were obtained and 58 samples suspicious of Shigella were specifically evaluated. The most prevalent isolated pathogen was Shigella sonnei (26/58) followed by E. coli (25/58) and Shigella flexneri (3/58). A large number of isolated bacteria were resistant to co-trimoxazole (Shigella spp: 100%, E. coli: 80%), azithromycin (Shigella spp: 70.4%, E. coli: 44.0%), ceftriaxone (Shigella spp: 88.9%, E. coli: 56.0%) and cefixime (Shigella spp: 85.2%, E. coli: 68.0%). About88.3% of S. sonnei isolates, one S. flexneri isolate, and 56% of E. coli strains were resistant to at least three antibiotic classes (multidrug resistant). Conclusion: Due to high levels of resistance to recommended and commonly used antibiotics for diarrhea, continuous monitoring of antibiotic resistance seems essential for determining best options of empirical therapy. PMID:25002896

  7. Virulence and antibiotic resistance of Escherichia coli isolated from rooks.

    PubMed

    Kmet, Vladimir; Drugdova, Zuzana; Kmetova, Marta; Stanko, Michal

    2013-01-01

    With regard to antibiotic resistance studies in various model animals in the urban environment, the presented study focused on the rook, many behavioural and ecological aspects of which are important from an epidemiological point of view. A total of 130 Escherichia coli strains isolated from rook faeces during a two-year period (2011-2012) were investigated for antibiotic resistance and virulence. Resistance to ampicillin (60%) and streptomycin (40%) were the most frequent, followed by resistance to fluoroquinolones (ciprofloxacin-22% and enrofloxacin-24%), tetracycline (18%), cotrimoxazol (17%) and florfenicol (14%). Ceftiofur resistance occured in 10.7% of strains and cefquinom resistance in 1.5% of strains. Twenty-five E.coli strains with a higher level of MICs of cephalosporins (over 2mg/L of ceftazidime and ceftriaxon) and fluoroquinolones were selected for detection of betalactamase genes (CTX-M, CMY), plasmid-mediated quinolone resistance qnrS, integrase 1, and for APEC (avian pathogenic E.coli) virulence factors (iutA, cvaC, iss, tsh, ibeA, papC, kpsII). Genes of CTX-M1, CMY-2, integrase 1, papC, cvaC, iutA were detected in one strain of E.coli, and qnrS, integrase 1, iss, cvaC, tsh were detected in another E.coli. DNA microarray revealed the absence of verotoxin and enterotoxin genes and pathogenicity islands. The results show that rooks can serve as a reservoir of antibiotic-resistant E. coli with avian pathogenic virulence factors for the human population, and potentially transmit such E.coli over long distances. PMID:23772573

  8. 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

  9. Non-O157 Shiga toxin-producing Escherichia coli: detection and characterization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli strains that produce Shiga toxins, referred to as Shiga toxin-producing E. coli (STEC) or verotoxigenic E. coli (VTEC) are important food-borne pathogens that cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). E. coli O157:H7 is a common cause of STEC infection; ho...

  10. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

    PubMed

    Njoroge, Samuel M; Boinett, Christine J; Madé, Laure F; Ouko, Tom T; Fèvre, Eric M; Thomson, Nicholas R; Kariuki, Samuel

    2015-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described. PMID:26187892

  11. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli

    PubMed Central

    Njoroge, Samuel M.; Boinett, Christine J.; Madé, Laure F.; Ouko, Tom T.; Fèvre, Eric M.; Thomson, Nicholas R.; Kariuki, Samuel

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described. PMID:26187892

  12. Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7▿

    PubMed Central

    Ruan, Lifang; Pleitner, Aaron; Gänzle, Michael G.; McMullen, Lynn M.

    2011-01-01

    This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50- to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins. PMID:21398480

  13. coliBASE: an online database for Escherichia coli, Shigella and Salmonella comparative genomics

    PubMed Central

    Chaudhuri, Roy R.; Khan, Arshad M.; Pallen, Mark J.

    2004-01-01

    We have constructed coliBASE, a database for Escherichia coli, Shigella and Salmonella comparative genomics available online at http://colibase.bham.ac.uk. Unlike other E.coli databases, which focus on the laboratory model strain K12, coliBASE is intended to reflect the full diversity of E.coli and its relatives. The database contains comparative data including whole genome alignments and lists of putative orthologous genes, together with numerous analytical tools and links to existing online resources. The data are stored in a relational database, accessible by a number of user-friendly search methods and graphical browsers. The database schema is generic and can easily be applied to other bacterial genomes. Two such databases, CampyDB (for the analysis of Campylobacter spp.) and ClostriDB (for Clostridium spp.) are also available at http://campy.bham.ac.uk and http://clostri.bham.ac.uk, respectively. An example of the power of E.coli comparative analyses such as those available through coliBASE is presented. PMID:14681417

  14. Development of functionalised polyelectrolyte capsules using filamentous Escherichia coli cells

    PubMed Central

    2012-01-01

    Background Escherichia coli is one of the best studied microorganisms and finds multiple applications especially as tool in the heterologous production of interesting proteins of other organisms. The heterologous expression of special surface (S-) layer proteins caused the formation of extremely long E. coli cells which leave transparent tubes when they divide into single E. coli cells. Such natural structures are of high value as bio-templates for the development of bio-inorganic composites for many applications. In this study we used genetically modified filamentous Escherichia coli cells as template for the design of polyelectrolyte tubes that can be used as carrier for functional molecules or particles. Diversity of structures of biogenic materials has the potential to be used to construct inorganic or polymeric superior hybrid materials that reflect the form of the bio-template. Such bio-inspired materials are of great interest in diverse scientific fields like Biology, Chemistry and Material Science and can find application for the construction of functional materials or the bio-inspired synthesis of inorganic nanoparticles. Results Genetically modified filamentous E. coli cells were fixed in 2% glutaraldehyde and coated with alternating six layers of the polyanion polyelectrolyte poly(sodium-4styrenesulfonate) (PSS) and polycation polyelectrolyte poly(allylamine-hydrochloride) (PAH). Afterwards we dissolved the E. coli cells with 1.2% sodium hypochlorite, thus obtaining hollow polyelectrolyte tubes of 0.7 μm in diameter and 5–50 μm in length. For functionalisation the polyelectrolyte tubes were coated with S-layer protein polymers followed by metallisation with Pd(0) particles. These assemblies were analysed with light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. Conclusion The thus constructed new material offers possibilities for diverse applications like novel catalysts or metal

  15. Determinants that increase the serum resistance of Escherichia coli.

    PubMed Central

    Taylor, P W; Robinson, M K

    1980-01-01

    The rfb locus, determining biosynthesis of O8-specific lipopolysaccharide side chains, was transferred to a rough mutant of Escherichia coli; recombinants producing a complete lipopolysaccharide were more resistant to the complement-mediated bactericidal action of human serum than the rough recipient. Inheritance of the his-linked genes for K27 antigen production did not alter the response to serum. The serum resistance of strains carrying O8 side chains, but not of strains with incomplete lipopolysaccharides, was further increased by inheritance of plasmids R1 and NR1.20 PMID:6995340

  16. 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

  17. An engineered eukaryotic protein glycosylation pathway in Escherichia coli.

    PubMed

    Valderrama-Rincon, Juan D; Fisher, Adam C; Merritt, Judith H; Fan, Yao-Yun; Reading, Craig A; Chhiba, Krishan; Heiss, Christian; Azadi, Parastoo; Aebi, Markus; DeLisa, Matthew P

    2012-05-01

    We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins. PMID:22446837

  18. Causes, prevention and treatment of Escherichia coli infections.

    PubMed

    Gould, Dinah

    Escherichia coli is a normal inhabitant of the human gastrointestinal tract and can cause healthcare-associated infections. The organism is most frequently responsible for urinary tract infections and it is the bacterium most often implicated in the cause of diarrhoea in people travelling overseas. In recent years, a strain called Ecoli O157 has gained notoriety for causing foodborne infection, which can have severe health consequences, especially in young children. This article describes the range of different infections caused by Ecoli in healthcare settings and the community and discusses the characteristics of the different strains of the bacteria that explain variations in their pathogenicity. PMID:20441035

  19. Molecular Evolution of the Escherichia Coli Chromosome. III. Clonal Frames

    PubMed Central

    Milkman, R.; Bridges, M. M.

    1990-01-01

    PCR fragments, 1500-bp, from 15 previously sequenced regions in the Escherichia coli chromosome have been compared by restriction analysis in a large set of wild (ECOR) strains. Prior published observations of segmental clonality are confirmed: each of several sequence types is shared by a number of strains. The rate of recombinational replacement and the average size of the replacements are estimated in a set of closely related strains in which a clonal frame is dotted with occasional stretches of DNA belonging to other clones. A clonal hierarchy is described. Some new comparative sequencing data are presented. PMID:1979037

  20. Global Properties of the Metabolic Map of Escherichia coli

    PubMed Central

    Ouzounis, Christos A.; Karp, Peter D.

    2000-01-01

    The EcoCyc database characterizes the known network of Escherichia coli small-molecule metabolism. Here we present a computational analysis of the global properties of that network, which consists of 744 reactions that are catalyzed by 607 enzymes. The reactions are organized into 131 pathways. Of the metabolic enzymes, 100 are multifunctional, and 68 of the reactions are catalyzed by >1 enzyme. The network contains 791 chemical substrates. Other properties considered by the analysis include the distribution of enzyme subunit organization, and the distribution of modulators of enzyme activity and of enzyme cofactors. The dimensions chosen for this analysis can be employed for comparative functional analysis of complete genomes. PMID:10779499

  1. Dual genetic selection of synthetic riboswitches in Escherichia coli.

    PubMed

    Nomura, Yoko; Yokobayashi, Yohei

    2014-01-01

    This chapter describes a genetic selection strategy to engineer synthetic riboswitches that can chemically regulate gene expression in Escherichia coli. Riboswitch libraries are constructed by randomizing the nucleotides that potentially comprise an expression platform and fused to the hybrid selection/screening marker tetA-gfpuv. Iterative ON and OFF selections are performed under appropriate conditions that favor the survival or the growth of the cells harboring the desired riboswitches. After the selection, rapid screening of individual riboswitch clones is performed by measuring GFPuv fluorescence without subcloning. This optimized dual genetic selection strategy can be used to rapidly develop synthetic riboswitches without detailed computational design or structural knowledge. PMID:24549616

  2. Biochemical and cultural characteristics of invasive Escherichia coli.

    PubMed Central

    Silva, R M; Toledo, M R; Trabulsi, L R

    1980-01-01

    The biochemical characteristics of 97 invasive Escherichia coli strains of different O serogroups were studied. Considered as a group, the behavior of the strains was quite variable. However, none of them decarboxylated lysine and all but seven strains, belonging to the O124 serogroup, were nonmotile. The growth of 25 strains obtained on MacConkey, salmonella-shigella, xylose-lysine-desoxycholate, and Hektoen enteric agars was compared. MacConkey and Hektoen enteric agars yielded the highest average growth for these strains, whereas salmonella-shigella agar had the lowest average counts. PMID:6991526

  3. 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. PMID:27026097

  4. Polarity effects in the lactose operon of Escherichia coli.

    PubMed

    Li, Yong; Altman, Sidney

    2004-05-21

    An intergenic RNA segment between lacY and lacA of the lactose operon in Escherichia coli is cleaved by RNase P, an endoribonuclease. The cleavage of the intergenic RNA was ten times less efficient than cleavage of a tRNA precursor in vitro. Fragments of the RNase P cleavage product are detectable in vivo in the wild-type strain but not in a mutant strain at the restrictive temperature. The cleavage product that contains lacA in the wild-type strain was quickly degraded. When this intergenic segment was cloned upstream of a reporter gene, the expression of the reporter gene was also inhibited substantially in wild-type E.coli, but not in a temperature sensitive mutant strain in RNase P at the restrictive temperature. These results support data regarding the natural polarity between lacZ versus lacA, the downstream gene. PMID:15123418

  5. Involvement of DNA superhelicity in minichromosome maintenance in Escherichia coli.

    PubMed Central

    Leonard, A C; Whitford, W G; Helmstetter, C E

    1985-01-01

    Evidence is presented that Escherichia coli minichromosomes are harbored at superhelical densities which are lower than those measured for other E. coli plasmids but are comparable to that of the chromosome. When introduced into gyrB decreased-supercoiling mutants, minichromosomes were much more unstable than in strains with normal or increased supercoiling properties; in fact, certain minichromosome derivatives could not be introduced into top gyrB decreased-supercoiling mutants. These observations were unique to minichromosomes, since the maintenance of plasmids which did not replicate from oriC was not altered in these mutants. Analyses of minichromosomes of identical sizes but with different restriction fragment orientations suggested that supercoiling-dependent alterations in promoter-terminator functions, as well as direct effects of supercoiling on replication, may play a role in the observed minichromosome instability. Images PMID:2981821

  6. Engineering Escherichia coli Cell Factories for n-Butanol Production.

    PubMed

    Dong, Hongjun; Zhao, Chunhua; Zhang, Tianrui; Lin, Zhao; Li, Yin; Zhang, Yanping

    2016-01-01

    The production of n-butanol, as a widely applied solvent and potential fuel, is attracting much attention. The fermentative production of butanol coupled with the production of acetone and ethanol by Clostridium (ABE fermentation) was once one of the oldest biotechnological processes, ranking second in scale behind ethanol fermentation. However, there remain problems with butanol production by Clostridium, especially the difficulty in genetically manipulating clostridial strains. In recent years, many efforts have been made to produce butanol using non-native strains. Until now, the most advanced effort was the engineering of the user-friendly and widely studied Escherichia coli for butanol production. This paper reviews the current progress and problems relating to butanol production by engineered E. coli in terms of prediction using mathematical models, pathway construction, novel enzyme replacement, butanol toxicity, and tolerance engineering strategies. PMID:25662903

  7. Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli

    PubMed Central

    Bury-Moné, Stéphanie; Nomane, Yanoura; Reymond, Nancie; Barbet, Romain; Jacquet, Eric; Imbeaud, Sandrine; Jacq, Annick; Bouloc, Philippe

    2009-01-01

    The Bae, Cpx, Psp, Rcs, and σE pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response. PMID:19763168

  8. The complete genome sequence of Escherichia coli K-12.

    PubMed

    Blattner, F R; Plunkett, G; Bloch, C A; Perna, N T; Burland, V; Riley, M; Collado-Vides, J; Glasner, J D; Rode, C K; Mayhew, G F; Gregor, J; Davis, N W; Kirkpatrick, H A; Goeden, M A; Rose, D J; Mau, B; Shao, Y

    1997-09-01

    The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer. PMID:9278503

  9. Studies on Resistance Transfer Factor Deoxyribonucleic Acid in Escherichia coli

    PubMed Central

    Silver, Richard P.; Falkow, Stanley

    1970-01-01

    A variant of the derepressed R factor, R1, which does not contain any of the drug resistance markers, and represents, in large part, the resistance transfer factor (RTF) was studied in Escherichia coli. RTF deoxyribonucleic acid (DNA) was specifically labeled in a female cell after conjugation. Physical characterization of the molecule showed that RTF possessed an average molecular weight of 50 × 106 daltons and a buoyant density of 1.709 g/cm3. By comparison to R1, we calculate that the region of DNA carrying the drug resistance genes is therefore about 20% of the R1 molecule and has a buoyant density of approximately 1.716 g/cm3. These results support the hypothesis that the single species of R-factor DNA observed in E. coli represents a composite of the 1.709 and 1.716 g/cm3 replicons seen in Proteus. PMID:4919749

  10. Two proline porters in Escherichia coli K-12.

    PubMed Central

    Stalmach, M E; Grothe, S; Wood, J M

    1983-01-01

    Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus. PMID:6355059

  11. Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli

    PubMed Central

    Mills, Tirzah Y; Sandoval, Nicholas R; Gill, Ryan T

    2009-01-01

    The sustainable production of biofuels will require the efficient utilization of lignocellulosic biomass. A key barrier involves the creation of growth-inhibitory compounds by chemical pretreatment steps, which ultimately reduce the efficiency of fermentative microbial biocatalysts. The primary toxins include organic acids, furan derivatives, and phenolic compounds. Weak acids enter the cell and dissociate, resulting in a drop in intracellular pH as well as various anion-specific effects on metabolism. Furan derivatives, dehydration products of hexose and pentose sugars, have been shown to hinder fermentative enzyme function. Phenolic compounds, formed from lignin, can disrupt membranes and are hypothesized to interfere with the function of intracellular hydrophobic targets. This review covers mechanisms of toxicity and tolerance for these compounds with a specific focus on the important industrial organism Escherichia coli. Recent efforts to engineer E. coli for improved tolerance to these toxins are also discussed. PMID:19832972

  12. Bleomycin Sensitivity in Escherichia coli is Medium-Dependent

    PubMed Central

    Xu, Tao; Brown, William; Marinus, Martin G.

    2012-01-01

    Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells. PMID:22438905

  13. 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. PMID:241475

  14. 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.

  15. Phenotypic and genotypic characterization of human and nonhuman Escherichia coli.

    PubMed

    Parveen, S; Hodge, N C; Stall, R E; Farrah, S R; Tamplin, M L

    2001-02-01

    Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates. PMID:11228989

  16. Strength and Regulation of Seven rRNA Promoters in Escherichia coli

    PubMed Central

    Maeda, Michihisa; Shimada, Tomohiro; Ishihama, Akira

    2015-01-01

    The model prokaryote Escherichia coli contains seven copies of the rRNA operon in the genome. The presence of multiple rRNA operons is an advantage for increasing the level of ribosome, the key apparatus of translation, in response to environmental conditions. The complete sequence of E. coli genome, however, indicated the micro heterogeneity between seven rRNA operons, raising the possibility in functional heterogeneity and/or differential mode of expression. The aim of this research is to determine the strength and regulation of the promoter of each rRNA operon in E. coli. For this purpose, we used the double-fluorescent protein reporter pBRP system that was developed for accurate and precise determination of the promoter strength of protein-coding genes. For application of this promoter assay vector for measurement of the rRNA operon promoters devoid of the signal for translation, a synthetic SD sequence was added at the initiation codon of the reporter GFP gene, and then approximately 500 bp-sequence upstream each 16S rRNA was inserted in front of this SD sequence. Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector. Results indicated that: the promoter activity was the highest for the rrnE promoter under all growth conditions analyzed, including different growth phases of wild-type E. coli grown in various media; but the promoter strength of other six rrn promoters was various depending on the culture conditions. These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations. Possible difference in the functional role of each rRNA operon is also discussed. PMID:26717514

  17. Chlamydial gene encoding a 70-kilodalton antigen in Escherichia coli: analysis of expression signals and identification of the gene product.

    PubMed Central

    Sardinia, L M; Engel, J N; Ganem, D

    1989-01-01

    In an attempt to identify chlamydial genes whose native promoters allow them to be expressed in Escherichia coli, we isolated and characterized a chlamydial gene identified by screening a library of chlamydial DNA with antichlamydial antibodies. This gene encodes a 70-kilodalton immunoreactive polypeptide in E. coli hosts. Sequence analysis of the 5' portion of the gene identified its product as the chlamydial homolog of the E. coli ribosomal protein S1. The site of transcription initiation of the mRNA in chlamydiae was determined, and its putative promoter regions were identified. These regions apparently do not function efficiently in E. coli; in vitro transcripts generated by using E. coli RNA polymerase did not start at the authentic chlamydial initiation site. Several in vitro transcripts both larger and smaller than the authentic transcript were seen; presumably, these transcripts result from adventitious promoterlike elements in adjacent chlamydial DNA and may be responsible for the expression of the gene in E. coli. Images PMID:2644193

  18. Genomic anatomy of Escherichia coli O157:H7 outbreaks.

    PubMed

    Eppinger, Mark; Mammel, Mark K; Leclerc, Joseph E; Ravel, Jacques; Cebula, Thomas A

    2011-12-13

    The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes. PMID:22135463

  19. Deactivation of Escherichia coli by the plasma needle

    NASA Astrophysics Data System (ADS)

    Sladek, R. E. J.; Stoffels, E.

    2005-06-01

    In this paper we present a parameter study on deactivation of Escherichia coli (E. coli) by means of a non-thermal plasma (plasma needle). The plasma needle is a small-sized (1 mm) atmospheric glow sustained by radio-frequency excitation. This plasma will be used to disinfect heat-sensitive objects; one of the intended applications is in vivo deactivation of dental bacteria: destruction of plaque and treatment of caries. We use E. coli films plated on agar dishes as a model system to optimize the conditions for bacterial destruction. Plasma power, treatment time and needle-to-sample distance are varied. Plasma treatment of E. coli films results in formation of a bacteria-free void with a size up to 12 mm. 104-105 colony forming units are already destroyed after 10 s of treatment. Prolongation of treatment time and usage of high powers do not significantly improve the destruction efficiency: short exposure at low plasma power is sufficient. Furthermore, we study the effects of temperature increase on the survival of E. coli and compare it with thermal effects of the plasma. The population of E. coli heated in a warm water bath starts to decrease at temperatures above 40°C. Sample temperature during plasma treatment has been monitored. The temperature can reach up to 60°C at high plasma powers and short needle-to-sample distances. However, thermal effects cannot account for bacterial destruction at low power conditions. For safe and efficient in vivo disinfection, the sample temperature should be kept low. Thus, plasma power and treatment time should not exceed 150 mW and 60 s, respectively.

  20. Emergence of imipenem resistance in clinical Escherichia coli during therapy.

    PubMed

    Oteo, Jesús; Delgado-Iribarren, Alberto; Vega, Dolores; Bautista, Verónica; Rodríguez, María Cruz; Velasco, María; Saavedra, José María; Pérez-Vázquez, María; García-Cobos, Silvia; Martínez-Martínez, Luis; Campos, José

    2008-12-01

    The molecular epidemiology and the mechanisms of resistance of Escherichia coli isolated from two patients infected by imipenem-resistant strains are reported in this study. From one patient, three closely related consecutive isolates of E. coli were recovered; the first was carbapenem-susceptible but acquired imipenem resistance after treatment with ertapenem, and the third isolate was again imipenem-susceptible. An additional imipenem-resistant isolate was recovered from another patient who received imipenem. The genetic relatedness of the E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE) after digestion with XbaI. Standard polymerase chain reaction (PCR) conditions were used to amplify several beta-lactamase genes coding for carbapenemases, extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC; the E. coli ampC gene promoter was also amplified and sequenced. Primers OmpF-F/OmpF-R and OmpC-F/OmpC-R were used to amplify the ompF and ompC genes. The outer membrane protein (OMP) profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Imipenem-resistant E. coli isolates did not produce carbapenemases but lacked the two major OMPs OmpF and OmpC and had ampC promoter mutations; in addition, one of the imipenem-resistant isolates produced the CMY-2 cephalosporinase, whilst the other produced the new CTX-M-67 ESBL. Carbapenem resistance in this study was associated with lack of expression of OmpF and OmpC porins. Additional mechanisms of beta-lactam resistance, such as plasmid-mediated AmpC and ESBL production, were also found. Development of carbapenem resistance in a CTX-M-67-producing E. coli is first described in this study. PMID:18775649

  1. Fecal leukocytes in children infected with diarrheagenic Escherichia coli.

    PubMed

    Mercado, Erik H; Ochoa, Theresa J; Ecker, Lucie; Cabello, Martin; Durand, David; Barletta, Francesca; Molina, Margarita; Gil, Ana I; Huicho, Luis; Lanata, Claudio F; Cleary, Thomas G

    2011-04-01

    The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response. PMID:21325554

  2. Dynamic Transcriptional Response of Escherichia coli to Inclusion Body Formation

    PubMed Central

    Baig, Faraz; Fernando, Lawrence P.; Salazar, Mary Alice; Powell, Rhonda R.; Bruce, Terri F.; Harcum, Sarah W.

    2014-01-01

    Escherichia coli is used intensively for recombinant protein production, but one key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously reported, the classical heat-shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis-related and energy-synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium. Additionally, putative genes represented over one-third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate that IB formation is complex, stimulates the heat-shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses. PMID:24338599

  3. Competition between congenic Escherichia coli K-12 strains in vivo.

    PubMed Central

    Onderdonk, A; Marshall, B; Cisneros, R; Levy, S B

    1981-01-01

    The ability of Escherichia coli to colonize the large bowels of animals is related to many factors inherent to the intestinal environment and the bacterium. The use of germfree mice eliminates the competition between E. coli and the other microflora and allows most E. coli strains to colonize. We found that E. coli K-12 strains differing in chromosomal antibiotic resistance could monoassociate in germfree mice in large numbers. However, when two or more strains were in competition with each other, we detected quantitative differences in the abilities of the strains to colonize. The order of colonizing ability was as follows: nalidixic acid resistance greater than streptomycin resistance greater than rifampin resistance. We also found that a nalidixic acid-resistant strain bearing plasmid pBR322 colonized less efficiently and at lower levels when in competition with the nalidixic acid-resistant strain. Studies of the membrane proteins of the various strains indicated that changes in membrane proteins occurred concomitantly with altered resistance to antimicrobial agents. These results suggest that chromosomally linked alterations in antimicrobial sensitivity may also reflect changes in membrane proteins and a decreased ability to colonize mammalian intestines in otherwise isogenic bacterial strains. Images PMID:7012037

  4. Modeling Escherichia coli removal in constructed wetlands under pulse loading.

    PubMed

    Hamaamin, Yaseen A; Adhikari, Umesh; Nejadhashemi, A Pouyan; Harrigan, Timothy; Reinhold, Dawn M

    2014-03-01

    Manure-borne pathogens are a threat to water quality and have resulted in disease outbreaks globally. Land application of livestock manure to croplands may result in pathogen transport through surface runoff and tile drains, eventually entering water bodies such as rivers and wetlands. The goal of this study was to develop a robust model for estimating the pathogen removal in surface flow wetlands under pulse loading conditions. A new modeling approach was used to describe Escherichia coli removal in pulse-loaded constructed wetlands using adaptive neuro-fuzzy inference systems (ANFIS). Several ANFIS models were developed and validated using experimental data under pulse loading over two seasons (winter and summer). In addition to ANFIS, a mechanistic fecal coliform removal model was validated using the same sets of experimental data. The results showed that the ANFIS model significantly improved the ability to describe the dynamics of E. coli removal under pulse loading. The mechanistic model performed poorly as demonstrated by lower coefficient of determination and higher root mean squared error compared to the ANFIS models. The E. coli concentrations corresponding to the inflection points on the tracer study were keys to improving the predictability of the E. coli removal model. PMID:24231031

  5. 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.

  6. Pathogenesis of Shiga-toxin producing escherichia coli.

    PubMed

    Melton-Celsa, Angela; Mohawk, Krystle; Teel, Louise; O'Brien, Alison

    2012-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) are food-borne pathogens that cause hemorrhagic colitis and a serious sequela, the hemolytic uremic syndrome (HUS). The largest outbreaks of STEC are due to a single E. coli serotype, O157:H7, although non-O157 serotypes also cause the same diseases. Two immunologically distinct Stxs are found in E. coli, Stx1 and Stx2. The Stxs are AB₅ toxins that halt protein synthesis in the host cell, a process that may lead to an apoptotic cell death. Stx-mediated damage to renal glomerular endothelial cells is hypothesized as the precipitating event for HUS. A subset of STEC referred to as the enterohemorrhagic E. coli has the capacity to intimately attach to and efface intestinal epithelial cells, a pathology called the A/E lesion. The A/E lesion is mediated by the adhesin intimin, its bacterially encoded receptor, Tir, and effectors secreted through a type III secretion system. The proteins needed for the A/E lesion are encoded within a large pathogenicity island called the locus of enterocyte effacement or LEE. There are several animal models for STEC infection, but no one model fully represents the spectrum of STEC illness. Currently there is no cure for STEC infection, and therapies are based mainly on alleviating symptoms. However, chimeric or humanized monoclonal antibodies have been developed that neutralize the Stxs, and those therapies may be able to prevent the development of HUS in an STEC-infected patient. PMID:21915773

  7. 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. PMID:23053089

  8. 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 ...

  9. Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli.

    PubMed

    Begam, Mehbuba; Kumar, Sushil; Roy, Sribash; Campanella, James J; Kapoor, H C

    2006-11-01

    A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV). PMID:16996549

  10. Mild gut inflammation modulates the proteome of intestinal Escherichia coli.

    PubMed

    Schumann, Sara; Alpert, Carl; Engst, Wolfram; Klopfleisch, Robert; Loh, Gunnar; Bleich, André; Blaut, Michael

    2014-09-01

    Using interleukin 10-deficient (IL-10(-/-) ) and wild-type mice monoassociated with either the adherent-invasive Escherichia coli UNC or the probiotic E. coli Nissle, the effect of a mild intestinal inflammation on the bacterial proteome was studied. Within 8 weeks, IL-10(-/-) mice monoassociated with E. coli UNC exhibited an increased expression of several proinflammatory markers in caecal mucosa. Escherichia coli Nissle-associated IL-10(-/-) mice did not do so. As observed previously for E. coli from mice with acute colitis, glycolytic enzymes were downregulated in intestinal E. coli UNC from IL-10(-/-) mice. In addition, the inhibitor of vertebrate C-type lysozyme, Ivy, was upregulated on messenger RNA (mRNA) and protein level in E. coli Nissle from IL-10(-/-) mice compared with E. coli UNC from these mice. Higher expression of Ivy in E. coli Nissle correlated with an improved growth of this probiotic strain in the presence of lysozyme-ethylenediaminetetraacetic acid (EDTA). By overexpressing Ivy, we demonstrated that Ivy contributes to a higher lysozyme resistance of E. coli, supporting the role of Ivy as a potential fitness factor. However, deletion of Ivy did not alter the growth phenotype of E. coli Nissle in the presence of lysozyme-EDTA, suggesting the existence of additional lysozyme inhibitors that can take over the function of Ivy. PMID:23855897

  11. Cleaving yeast and Escherichia coli genomes at a single site

    SciTech Connect

    Koob, M.; Szybalski, W. )

    1990-10-12

    The 15-megabase pair Saccharomyces cerevisiae and the 4.7-megabase pair Escherichia coli genomes were completely cleaved at a single predetermined site by means of the Achilles' heel cleavage (AC) procedure. The symmetric lac operator (lacO{sub s}) was introduced into the circular Escherichia coli genome and into one of the 16 yeast chromosomes. Intact chromosomes from the resulting strains were prepared in agarose microbeads and methylated with Hha I (5{prime}-GCGC) methyltransferase (M{center dot}Hha I) in the presence of lac repressor (LacI). All Hae II sites ({prime}-{sub G}{sup A}GCGC{sub C}{sup T}) with the exception of the one in lacO{sub s}, which was protected by LacI, were modified and thus no longer recognized by Hae II. After inactivation of M{center dot}Hha I and LacI, Hae II was used to completely cleave the chromosomes specifically at the inserted lacO{sub s}. These experiments demonstrate the feasibility of using the AC approach to efficiently extend the specificity of naturally occurring restriction enzymes and create new tools for the mapping and precise molecular dissection of multimegabase genomes.

  12. Induction of SOS genes of Escherichia coli by chromium compounds

    SciTech Connect

    Llagostera, M.; Garrido, S.; Guerrero, R.; Barbe, J.

    1986-01-01

    The induction of several SOS genes of Escherichia coli such as recA, umuC, and sfiA by hexavalent (K/sub 2/Cr/sub 2/O/sub 7/, K/sub 2/CrO/sub 4/, and CrO/sub 3/) and trivalent (CrCl/sub 3/, Cr(NO/sub 3/)/sub 3/, and (CH/sub 3/COO)/sub 3/Cr) compounds of chromium was studied. Induction was measured as ..beta..-galactosidase activity, using lacZ gene fusions under the control region of different SOS genes. The hexavalent chromium forms induced the genes responsible for massive synthesis of RecA protein, error-prone repair, and inhibition of cell division. On the other hand, the trivalent chromium compounds were unable to induce any of the SOS genes tested. Individual assay of hexavalent chromium compounds showed that K/sub 2/Cr/sub 2/O/sub 7/ was a stronger inducing agent of those three SOS genes tested than K/sub 2/CrO/sub 4/, which, in turn, was stronger than CrO/sub 3/. All this data led to the conclusion that hexavalent chromium compounds, but not trivalent, are proficient agents of induction of the SOS system and can produce indirect mutagenesis in Escherichia coli.

  13. The Model [NiFe]-Hydrogenases of Escherichia coli.

    PubMed

    Sargent, F

    2016-01-01

    In Escherichia coli, hydrogen metabolism plays a prominent role in anaerobic physiology. The genome contains the capability to produce and assemble up to four [NiFe]-hydrogenases, each of which are known, or predicted, to contribute to different aspects of cellular metabolism. In recent years, there have been major advances in the understanding of the structure, function, and roles of the E. coli [NiFe]-hydrogenases. The membrane-bound, periplasmically oriented, respiratory Hyd-1 isoenzyme has become one of the most important paradigm systems for understanding an important class of oxygen-tolerant enzymes, as well as providing key information on the mechanism of hydrogen activation per se. The membrane-bound, periplasmically oriented, Hyd-2 isoenzyme has emerged as an unusual, bidirectional redox valve able to link hydrogen oxidation to quinone reduction during anaerobic respiration, or to allow disposal of excess reducing equivalents as hydrogen gas. The membrane-bound, cytoplasmically oriented, Hyd-3 isoenzyme is part of the formate hydrogenlyase complex, which acts to detoxify excess formic acid under anaerobic fermentative conditions and is geared towards hydrogen production under those conditions. Sequence identity between some Hyd-3 subunits and those of the respiratory NADH dehydrogenases has led to hypotheses that the activity of this isoenzyme may be tightly coupled to the formation of transmembrane ion gradients. Finally, the E. coli genome encodes a homologue of Hyd-3, termed Hyd-4, however strong evidence for a physiological role for E. coli Hyd-4 remains elusive. In this review, the versatile hydrogen metabolism of E. coli will be discussed and the roles and potential applications of the spectrum of different types of [NiFe]-hydrogenases available will be explored. PMID:27134027

  14. 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

  15. Escherichia coli ghosts promote innate immune responses in human keratinocytes.

    PubMed

    Abtin, Arby; Kudela, Pavol; Mayr, Ulrike Beate; Koller, Verena Juliana; Mildner, Michael; Tschachler, Erwin; Lubitz, Werner

    2010-09-10

    Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin. PMID:20696136

  16. 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

  17. 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

  18. Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli.

    PubMed

    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

  19. Fast-tumbling bicelles constructed from native Escherichia coli lipids.

    PubMed

    Liebau, Jobst; Pettersson, Pontus; Zuber, Philipp; Ariöz, Candan; Mäler, Lena

    2016-09-01

    Solution-state NMR requires small membrane mimetic systems to allow for acquiring high-resolution data. At the same time these mimetics should faithfully mimic biological membranes. Here we characterized two novel fast-tumbling bicelle systems with lipids from two Escherichia coli strains. While strain 1 (AD93WT) contains a characteristic E. coli lipid composition, strain 2 (AD93-PE) is not capable of synthesizing the most abundant lipid in E. coli, phosphatidylethanolamine. The lipid and acyl chain compositions were characterized by (31)P and (13)C NMR. Depending on growth temperature and phase, the lipid composition varies substantially, which means that the bicelle composition can be tuned by using lipids from cells grown at different temperatures and growth phases. The hydrodynamic radii of the bicelles were determined from translational diffusion coefficients and NMR spin relaxation was measured to investigate lipid properties in the bicelles. We find that the lipid dynamics are unaffected by variations in lipid composition, suggesting that the bilayer is in a fluid phase under all conditions investigated here. Backbone glycerol carbons are the most rigid positions in all lipids, while head-group carbons and the first carbons of the acyl chain are somewhat more flexible. The flexibility increases down the acyl chain to almost unrestricted motion at its end. Carbons in double bonds and cyclopropane moieties are substantially restricted in their motional freedom. The bicelle systems characterized here are thus found to faithfully mimic E. coli inner membranes and are therefore useful for membrane interaction studies of proteins with E. coli inner membranes by solution-state NMR. PMID:27317394

  20. 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...

  1. 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...

  2. 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...

  3. 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

  4. 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. PMID:27332118

  5. Characterization of the YdeO Regulon in Escherichia coli

    PubMed Central

    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. PMID:25375160

  6. 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. PMID:26435280

  7. Pet, an Autotransporter Enterotoxin from Enteroaggregative Escherichia coli

    PubMed Central

    Eslava, Carlos; Navarro-García, Fernando; Czeczulin, John R.; Henderson, Ian R.; Cravioto, Alejandro; Nataro, James P.

    1998-01-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenic E. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects. PMID:9632580

  8. Shiga Toxin (Verotoxin)-Producing Escherichia coli in Japan.

    PubMed

    Terajima, Jun; Iyoda, Sunao; Ohnishi, Makoto; Watanabe, Haruo

    2014-10-01

    A series of outbreaks of infection with Shiga toxin (verocytotoxin)-producing Escherichia coli or enterohemorrhagic E. coli (EHEC) O157:H7 occurred in Japan in 1996, the largest outbreak occurring in primary schools in Sakai City, Osaka Prefecture, where more than 7,500 cases were reported. Although the reason for the sudden increase in the number of reports of EHEC isolates in 1996 is not known, the number of reports has grown to more than 3,000 cases per year since 1996, from an average of 105 reports each year during the previous 5-year period (1991-1995). Despite control measures instituted since 1996, including designating Shiga toxin-producing E. coli infection as a notifiable disease, and nationwide surveillance effectively monitoring the disease, the number of reports remains high, around 3,800 cases per year. Serogroup O157 predominates over other EHEC serogroups, but isolation frequency of non-O157 EHEC has gone up slightly over the past few years. Non-O157 EHEC has recently caused outbreaks where consumption of a raw beef dish was the source of the infection, and some fatal cases occurred. Laboratory surveillance comprised prefectural and municipal public health institutes, and the National Institute of Infectious Diseases has contributed to finding not only multiprefectural outbreaks but recognizing sporadic cases that could have been missed as an outbreak without the aid of molecular subtyping of EHEC isolates. This short overview presents recent information on the surveillance of EHEC infections in Japan. PMID:26104366

  9. Metabolic Engineering of Escherichia coli for the Production of Xylonate

    PubMed Central

    Cao, Yujin; Xian, Mo; Zou, Huibin; Zhang, Haibo

    2013-01-01

    Xylonate is a valuable chemical for versatile applications. Although the chemical synthesis route and microbial conversion pathway were established decades ago, no commercial production of xylonate has been obtained so far. In this study, the industrially important microorganism Escherichia coli was engineered to produce xylonate from xylose. Through the coexpression of a xylose dehydrogenase (xdh) and a xylonolactonase (xylC) from Caulobacter crescentus, the recombinant strain could convert 1 g/L xylose to 0.84 g/L xylonate and 0.10 g/L xylonolactone after being induced for 12 h. Furthermore, the competitive pathway for xylose catabolism in E. coli was blocked by disrupting two genes (xylA and xylB) encoding xylose isomerase and xylulose kinase. Under fed-batch conditions, the finally engineered strain produced up to 27.3 g/L xylonate and 1.7 g/L xylonolactone from 30 g/L xylose, about 88% of the theoretical yield. These results suggest that the engineered E. coli strain has a promising perspective for large-scale production of xylonate. PMID:23861757

  10. Escherichia coli lipoprotein binds human plasminogen via an intramolecular domain

    PubMed Central

    Gonzalez, Tammy; Gaultney, Robert A.; Floden, Angela M.; Brissette, Catherine A.

    2015-01-01

    Escherichia coli lipoprotein (Lpp) is a major cellular component that exists in two distinct states, bound-form and free-form. Bound-form Lpp is known to interact with the periplasmic bacterial cell wall, while free-form Lpp is localized to the bacterial cell surface. A function for surface-exposed Lpp has yet to be determined. We hypothesized that the presence of C-terminal lysinses in the surface-exposed region of Lpp would facilitate binding to the host zymogen plasminogen (Plg), a protease commandeered by a number of clinically important bacteria. Recombinant Lpp was synthesized and the binding of Lpp to Plg, the effect of various inhibitors on this binding, and the effects of various mutations of Lpp on Lpp–Plg interactions were examined. Additionally, the ability of Lpp-bound Plg to be converted to active plasmin was analyzed. We determined that Lpp binds Plg via an atypical domain located near the center of mature Lpp that may not be exposed on the surface of intact E. coli according to the current localization model. Finally, we found that Plg bound by Lpp can be converted to active plasmin. While the consequences of Lpp binding Plg are unclear, these results prompt further investigation of the ability of surface exposed Lpp to interact with host molecules such as extracellular matrix components and complement regulators, and the role of these interactions in infections caused by E. coli and other bacteria. PMID:26500634

  11. Metabolomic and transcriptomic stress response of Escherichia coli

    PubMed Central

    Jozefczuk, Szymon; Klie, Sebastian; Catchpole, Gareth; Szymanski, Jedrzej; Cuadros-Inostroza, Alvaro; Steinhauser, Dirk; Selbig, Joachim; Willmitzer, Lothar

    2010-01-01

    Environmental fluctuations lead to a rapid adjustment of the physiology of Escherichia coli, necessitating changes on every level of the underlying cellular and molecular network. Thus far, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. Here, we incorporate the metabolite composition together with gene expression data to provide a more comprehensive insight on system level stress adjustments by describing detailed time-resolved E. coli response to five different perturbations (cold, heat, oxidative stress, lactose diauxie, and stationary phase). The metabolite response is more specific as compared with the general response observed on the transcript level and is reflected by much higher specificity during the early stress adaptation phase and when comparing the stationary phase response to other perturbations. Despite these differences, the response on both levels still follows the same dynamics and general strategy of energy conservation as reflected by rapid decrease of central carbon metabolism intermediates coinciding with downregulation of genes related to cell growth. Application of co-clustering and canonical correlation analysis on combined metabolite and transcript data identified a number of significant condition-dependent associations between metabolites and transcripts. The results confirm and extend existing models about co-regulation between gene expression and metabolites demonstrating the power of integrated systems oriented analysis. PMID:20461071

  12. Biofilm and fluoroquinolone resistance of canine Escherichia coli uropathogenic isolates

    PubMed Central

    2014-01-01

    Background Escherichia coli is the most common uropathogen involved in urinary tract infection (UTI). Virulence of strains may differ, and may be enhanced by antimicrobial resistance and biofilm formation, resulting in increased morbidity and recurrent infections. The aim of this study was to evaluate the in vitro biofilm forming capacity of E. coli isolates from dogs with UTI, by using fluorescent in situ hybridization, and its association with virulence genes and antimicrobial resistance. Findings The proportion of biofilm-producing isolates significantly increased with the length of incubation time (P < 0.05). Biofilm production was significantly associated with fluoroquinolone resistance at all incubation time points and was independent of the media used (P < 0.05). Biofilm production was not associated with cnf1, hly, pap and sfa genes (P > 0.05), but was significantly associated with afa, aer and the β-lactamase genes (P < 0.05). Conclusions To the best of our knowledge, this is the first report showing significant association between biofilm production and fluoroquinolone resistance in E. coli isolates from dogs with UTI. Biofilm formation may contribute to UTI treatment failure in dogs, through the development of bacterial reservoirs inside bladder cells, allowing them to overcome host immune defenses and to establish recurrent infections. PMID:25099929

  13. Lactobacillus prophylaxis for diarrhea due to enterotoxigenic Escherichia coli.

    PubMed Central

    Clements, M L; Levine, M M; Black, R E; Robins-Browne, R M; Cisneros, L A; Drusano, G L; Lanata, C F; Saah, A J

    1981-01-01

    In vitro and animal experiments indicated that lactobacilli might prevent Escherichia coli from colonizing the intestine and may produce substances counteracting enterotoxin. Lactinex, a commercial preparation of dried Lactobacillus acidophilus and L. bulgaricus, is marketed for uncomplicated diarrhea. Preliminary experiments in nonfasting volunteers indicated that lactobacilli in this preparation colonized the small intestine for up to 6 h. To evaluate the protective efficacy of Lactinex, a double-blind randomized study was carried out in which 48 volunteers (23 receiving Lactinex and 25 receiving placebos) were challenged with E. coli strains that produced heat-stable or heat-labile enterotoxins or both. No significant differences between the two groups were noted with respect to attack rate, incubation period, duration of diarrhea, volume and number of liquid stools, and coproculture yields. These data suggest that this lactobacillus preparations does not prevent or alter the course of enterotoxigenic E. coli diarrhea in adults. Lack of efficacy occurred despite efforts to maximize small bowel colonization, including administration of Lactinex in milk and in a 6-hour-interval regimen during 36 h before and 96 h after challenge. PMID:6792978

  14. Thiols, recA induction and radiosensitivity in Escherichia coli.

    PubMed

    Naslund, M; Anderstam, B; Granath, F; Ehrenberg, L

    1996-01-01

    Induction by gamma-radiation, UV radiation or hydroxyurea of RecA gene product synthesis in Escherichia coli, monitored as beta-D-galactosidase in recA-lacZ fusion strains, was shown to be inhibited if 2-mercaptoethylamine (MEA) was added before treatment with the inducing agents. If cysteine (Cys) at low concentrations was added at the same time as MEA it counteracted the action of MEA. The effect of MEA may be described as a competitive inhibition of an inducing or conducting effect of Cys. In E. coli GE499 (uvrA+), complete inhibition by 30-mmol dm-3 MEA of recA induction was associated with about five times higher radio-resistence. Both of these effects of MEA were completely reversed by 0.3-mmol dm-3 Cys. As shown in parallel experiments with E. coli GE500 (uvrA-), these effects of MEA and Cys were shown to be independent of excision-repair proficiency. Treatment of bacteria with MEA and/or Cys was shown not to lead to increased intracellular concentrations of these thiols. Instead, treatment with them appeared to provoke conspicuous increases in glutathione levels, which are, however, probably not directly involved in the studied action of MEA and Cys. PMID:8601760

  15. Discrepancies in the Enumeration of Escherichia coli1

    PubMed Central

    Ray, B.; Speck, M. L.

    1973-01-01

    Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO4.7H2O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods. PMID:4572980

  16. 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. PMID:26476644

  17. Metabolic engineering of Escherichia coli to produce zeaxanthin.

    PubMed

    Li, Xi-Ran; Tian, Gui-Qiao; Shen, Hong-Jie; Liu, Jian-Zhong

    2015-04-01

    Zeaxanthin is a high-value carotenoid that is used in nutraceuticals, cosmetics, food, and animal feed industries. Zeaxanthin is chemically synthesized or purified from microorganisms as a natural product; however, increasing demand requires development of alternative sources such as heterologous biosynthesis by recombinant bacteria. For this purpose, we molecularly engineered Escherichia coli to optimize the synthesis of zeaxanthin from lycopene using fusion protein-mediated substrate channeling as well as by the introduction of tunable intergenic regions. The tunable intergenic regions approach was more efficient compared with protein fusion for coordinating expression of lycopene β-cyclase gene crtY and β-carotene 3-hydroxylase gene crtZ. The influence of the substrate channeling effect suggests that the reaction catalyzed by CrtZ is the rate-limiting step in zeaxanthin biosynthesis. Then Pantoea ananatis, Pantoea agglomerans and Haematococcus pluvialis crtZ were compared. Because P. ananatis crtZ is superior to that of P. agglomerans or H. pluvialis for zeaxanthin production, we used it to generate a recombinant strain of E. coli BETA-1 containing pZSPBA-2(P37-crtZPAN) that produced higher amounts of zeaxanthin (11.95 ± 0.21 mg/g dry cell weight) than other engineered E. coli strains described in the literature. PMID:25533633

  18. Binding of type 1-piliated Escherichia coli to vaginal mucus.

    PubMed Central

    Venegas, M F; Navas, E L; Gaffney, R A; Duncan, J L; Anderson, B E; Schaeffer, A J

    1995-01-01

    To better understand the interactions involved in bacterial adherence and the role of mucus in the pathogenesis of urinary tract infections, we developed a system to study the binding of a recombinant Escherichia coli strain, HB101/pWRS1-17, expressing type 1 pili, to vaginal mucus collected from 28 women. Bacteria bound to differing extents to all specimens examined, and preincubation of bacteria with mannose inhibited binding by 50 to 89%. Additionally, all mucus samples showed reactivity with anti-mannose antibody, and the levels of reactivity correlated with the levels of bacterial binding, suggesting that the mannose-terminal saccharides present on these glycoproteins are the receptors for the binding of type 1-piliated bacteria. Mucus specimens collected over periods of 5 days and 12 weeks exhibited significant variation in bacterial binding, indicating temporal differences in the ability of vaginal mucus to act as a receptor for type 1-piliated E. coli. The results show that vaginal mucus can bind bacteria and may thus influence the initial attachment and subsequent colonization of the vaginal and urinary tract epithelium by E. coli. PMID:7822005

  19. 2DBase: 2D-PAGE database of Escherichia coli.

    PubMed

    Vijayendran, Chandran; Burgemeister, Sebastian; Friehs, Karl; Niehaus, Karsten; Flaschel, Erwin

    2007-11-23

    We present a web-based integrated proteome database, termed 2DBase of Escherichia coli which was designed to store, compare, analyse, and retrieve various information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry. The main objectives of this database are (1) to provide the features for query and data-mining applications to access the stored proteomics data (2) to efficiently compare the specific protein spots present in the comparable proteome maps and (3) to analyse the data with the integrated classification for cellular functions of gene products of E. coli. This database currently contains 12 gels consisting of 1185 protein spots information in which 723 proteins were identified and annotated. Individual protein spots in the existing gels can be displayed, queried, analyzed, and compared in a tabular format based on various functional categories enabling quick and subsequent analyses. Our database satisfies the requirement to be a federated 2-DE database by accomplishing various tasks through a web interface providing access to a relational database system. The 2DBase of E. coli database can be accessed at http://2dbase.techfak.uni-bielefeld.de/. PMID:17904107

  20. 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

  1. Reduction of aerobic acetate production by Escherichia coli.

    PubMed Central

    Farmer, W R; Liao, J C

    1997-01-01

    Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose. PMID:9251207

  2. Epidemiological studies of congo red Escherichia coli in broiler chickens.

    PubMed Central

    Stebbins, M E; Berkhoff, H A; Corbett, W T

    1992-01-01

    This prospective cohort study was designed to confirm the association between Congo red binding Escherichia coli (CREC) and E. coli air sacculitis in commercial broilers. It was also designed to evaluate CREC as an air sacculitis risk factor and to explore the CREC relationship to other air sacculitis risk factors (poultry house temperature, air-ammonia levels, and presence of other diseases). In addition, this study was used to assess a possible role of the broiler-breeder flocks and hatchers in the spread of CREC air sacculitis. Congo red E. coli-associated airsacculitis risk was based on CREC exposure of the chicks in the hatchers. Breeder flocks with greater than 30 CREC colonies/plate from hatcher air sampling tests were placed in the high risk group; flocks with less than five CREC colonies/plate were placed in the low risk group. Increased risks of death due to air sacculitis (RR = 2.26), and increased death rates due to CREC air sacculitis (RR = 9.45) in high-risk flocks, identified CREC as an important air sacculitis risk factor. The attributable risk percent of CREC airsacculitis from hatcher exposure of CREC was 89.4%, pointing to the hatcher as the source of CREC infection. The association of specific broiler-breeder flocks to high levels of CREC in the hatchers, and subsequent air sacculitis, suggests that the broiler-breeders are the ultimate source of CREC. PMID:1423058

  3. Survival of Escherichia coli and Salmonella spp. in estuarine environments.

    PubMed

    Rhodes, M W; Kator, H

    1988-12-01

    Survival of Escherichia coli and Salmonella spp. in estuarine waters was compared over a variety of seasonal temperatures during in situ exposure in diffusion chambers. Sublethal stress was measured by both selective-versus-resuscitative enumeration procedures and an electrochemical detection method. E. coli and Salmonella spp. test suspensions, prepared to minimize sublethal injury, were exposed in a shallow tidal creek and at a site 7.1 km further downriver. Bacterial die-off and sublethal stress in filtered estuarine water were inversely related to water temperature. Salmonella spp. populations exhibited significantly less die-off and stress than did E. coli at water temperatures of less than 10 degrees C. Although the most pronounced reductions (ca. 3 log units) in test bacteria occurred during seasonally warm temperatures in the presence of the autochthonous microbiota, 10(2) to 10(4) test cells per ml remained after 2 weeks of exposure to temperatures of greater than 15 degrees C. Reductions in test bacteria were associated with increases in the densities of microflagellates and plaque-forming microorganisms. These studies demonstrated the survival potential of enteric bacteria in estuarine waters and showed that survival was a function of interacting biological and physical factors. PMID:3066291

  4. 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. PMID:18758769

  5. 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

  6. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli.

    PubMed

    Kay, Emily J; Yates, Laura E; Terra, Vanessa S; Cuccui, Jon; Wren, Brendan W

    2016-04-01

    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

  7. Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

    PubMed Central

    Bleibtreu, Alexandre; Gros, Pierre-Alexis; Laouénan, Cédric; Clermont, Olivier; Le Nagard, Hervé; Picard, Bertrand; Tenaillon, Olivier

    2013-01-01

    The extraintestinal virulence of Escherichia coli is dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuring in vitro individual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenic E. coli strains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescent E. coli strain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H2O2 resistance, and rpoS sequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains. PMID:23690401

  8. Neisseria gonorrhoeae prepilin export studied in Escherichia coli.

    PubMed Central

    Dupuy, B; Taha, M K; Pugsley, A P; Marchal, C

    1991-01-01

    The pilE gene of Neisseria gonorrhoeae MS11 and a series of pilE-phoA gene fusions were expressed in Escherichia coli. The PhoA hybrid proteins were shown to be located in the membrane fraction of the cells, and the prepilin product of the pilE gene was shown to be located exclusively in the cytoplasmic membrane. Analysis of the prepilin-PhoA hybrids showed that the first 20 residues of prepilin can function as an efficient export (signal) sequence. This segment of prepilin includes an unbroken sequence of 8 hydrophobic or neutral residues that form the N-terminal half of a 16-residue hydrophobic region of prepilin. Neither prepilin nor the prepilin-PhoA hybrids were processed by E. coli leader peptidase despite the presence of two consensus cleavage sites for this enzyme just after this hydrophobic region. Comparisons of the specific molecular activities of the four prepilin-PhoA hybrids and analysis of their susceptibility to proteolysis by trypsin and proteinase K in spheroplasts allow us to propose two models for the topology of prepilin in the E. coli cytoplasmic membrane. The bulk of the evidence supports the simplest of the two models, in which prepilin is anchored in the membrane solely by the N-terminal hydrophobic domain, with the extreme N terminus facing the cytoplasm and the longer C terminus facing the periplasm. Images FIG. 2 FIG. 4 FIG. 5 FIG. 6 PMID:1938955

  9. Starved Escherichia coli preserve reducing power under nitric oxide stress.

    PubMed

    Gowers, Glen-Oliver F; Robinson, Jonathan L; Brynildsen, Mark P

    2016-07-15

    Nitric oxide (NO) detoxification enzymes, such as NO dioxygenase (NOD) and NO reductase (NOR), are important to the virulence of numerous bacteria. Pathogens use these defense systems to ward off immune-generated NO, and they do so in environments that contain additional stressors, such as reactive oxygen species, nutrient deprivation, and acid stress. NOD and NOR both use reducing equivalents to metabolically deactivate NO, which suggests that nutrient deprivation could negatively impact their functionality. To explore the relationship between NO detoxification and nutrient deprivation, we examined the ability of Escherichia coli to detoxify NO under different levels of carbon source availability in aerobic cultures. We observed failure of NO detoxification under both carbon source limitation and starvation, and those failures could have arisen from inabilities to synthesize Hmp (NOD of E. coli) and/or supply it with sufficient NADH (preferred electron donor). We found that when limited quantities of carbon source were provided, NO detoxification failed due to insufficient NADH, whereas starvation prevented Hmp synthesis, which enabled cells to maintain their NADH levels. This maintenance of NADH levels under starvation was confirmed to be dependent on the absence of Hmp. Intriguingly, these data show that under NO stress, carbon-starved E. coli are better positioned with regard to reducing power to cope with other stresses than cells that had consumed an exhaustible amount of carbon. PMID:27207837

  10. [Improving 3-dehydroshikimate production by metabolically engineered Escherichia coli].

    PubMed

    Yuan, Fei; Chen, Wujiu; Jia, Shiru; Wang, Qinhong

    2014-10-01

    In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS. PMID:25726580

  11. Colibri: a functional data base for the Escherichia coli genome.

    PubMed Central

    Médigue, C; Viari, A; Hénaut, A; Danchin, A

    1993-01-01

    Several data libraries have been created to organize all the data obtained worldwide about the Escherichia coli genome. Because the known data now amount to more than 40% of the whole genome sequence, it has become necessary to organize the data in such a way that appropriate procedures can associate knowledge produced by experiments about each gene to its position on the chromosome and its relation to other relevant genes, for example. In addition, global properties of genes, affected by the introduction of new entries, should be present as appropriate description fields. A data base, implemented on Macintosh by using the data base management system 4th Dimension, is described. It is constructed around a core constituted by known contigs of E. coli sequences and links data collected in general libraries (unmodified) to data associated with evolving knowledge (with modifiable fields). Biologically significant results obtained through the coupling of appropriate procedures (learning or statistical data analysis) are presented. The data base is available through a 4th Dimension runtime and through FTP on Internet. It has been regularly updated and will be systematically linked to other E. coli data bases (M. Kroger, R. Wahl, G. Schachtel, and P. Rice, Nucleic Acids Res. 20(Suppl.):2119-2144, 1992; K. E. Rudd, W. Miller, C. Werner, J. Ostell, C. Tolstoshev, and S. G. Satterfield, Nucleic Acids Res. 19:637-647, 1991) in the near future. Images PMID:8246843

  12. Temperature Control of Phospholipid Biosynthesis in Escherichia coli

    PubMed Central

    Sinensky, Michael

    1971-01-01

    The higher the growth temperature of Escherichia coli cultures the greater is the proportion of saturated fatty acids in the bacterial phospholipids. When fatty acids are exogenously supplied to E. coli, higher growth temperatures will likewise increase the relative incorporation of saturated fatty acids into phospholipids. One of the steps in the utilization of fatty acids for phospholipid biosynthesis is, therefore, temperature-controlled. The temperature effect observed in vivo with mixtures of 3H-oleate and 14C-palmitate is demonstrable in vitro by using mixtures of the coenzyme A derivative of these fatty acids for the acylation of α-glycerol phosphate to lysophosphatidic and phosphatidic acids. In E. coli extracts, the relative rates of transacylation of palmityl and oleyl coenzyme A vary as a function of incubation temperature in a manner which mimics the temperature control observed in vivo. The phosphatidic acid synthesized in vitro shows a striking enrichment of oleate at the β position analogous to the positional specificity observed in phospholipids synthesized in vivo. PMID:4324806

  13. 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

  14. Protein secretion controlled by a synthetic gene in Escherichia coli.

    PubMed

    Blanchin-Roland, S; Masson, J M

    1989-03-01

    The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain. PMID:2652141

  15. 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

  16. Biofilm formation by Escherichia coli in hypertonic sucrose media.

    PubMed

    Kawarai, Taketo; Furukawa, Soichi; Narisawa, Naoki; Hagiwara, Chisato; Ogihara, Hirokazu; Yamasaki, Makari

    2009-06-01

    High osmotic environments produced by NaCl or sucrose have been used as reliable and traditional methods of food preservation. We tested, Escherichia coli as an indicator of food-contaminating bacterium, to determine if it can form biofilm in a hyperosmotic environment. E. coli K-12 IAM1264 did not form biofilm in LB broth that contained 1 M NaCl. However, the bacterium formed biofilm in LB broth that contained 1 M sucrose, although the planktonic growth was greatly suppressed. The biofilm, formed on solid surfaces, such as titer-plate well walls and glass slides, solely around the air-liquid interface. Both biofilm forming cells and planktonic cells in the hypertonic medium adopted a characteristic, fat and filamentous morphology with no FtsZ rings, which are a prerequisite for septum formation. Biofilm forming cells were found to be alive based on propidium iodide staining. The presence of 1 M sucrose in the food environment is not sufficient to prevent biofilm formation by E. coli. PMID:19447340

  17. Epithelial cell invasion by bovine septicemic Escherichia coli.

    PubMed Central

    Korth, M J; Lara, J C; Moseley, S L

    1994-01-01

    Little is known regarding the pathogenesis of Escherichia coli-induced septicemic colibacillosis of calves. To understand the mechanism by which these strains penetrate the intestinal epithelium and gain access to the bloodstream, we examined the potential of bovine septicemic E. coli to invade cultured epithelial cells. By using a gentamicin survival assay, we demonstrated bacterial invasion of Madin-Darby canine kidney (MDCK) cells. Transcytosis of polarized MDCK cell monolayers was also observed, but only when bacteria were added to the basolateral surface. Electron microscopy confirmed the presence of intracellular organisms which appeared to be within membrane-bound vacuoles. The bovine septicemic isolate used in this study expressed the fimbrial adhesion CS31A. To examine the role of CS31A-mediated adherence in invasion and transcytosis of MDCK cell monolayers, a CS31A-deficient mutant was constructed by suicide vector-mediated insertional mutagenesis. Although nonadherent, the mutant showed a level of invasion similar to that of the wild-type parent. E. coli DH5 alpha carrying the cloned CS31A determinant was noninvasive. These findings suggest that expression of CS31A is neither required nor sufficient to mediate invasion. Images PMID:7903284

  18. Properties of a Clostridium thermocellum Endoglucanase Produced in Escherichia coli.

    PubMed

    Schwarz, W H; Gräbnitz, F; Staudenbauer, W L

    1986-06-01

    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-beta-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 degrees C and was stable for several hours at 60 degrees 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 beta-1,4 and beta-1,3 linkages such as barley beta-glucan and lichenan. PMID:16347088

  19. Rapid Method for Escherichia coli in the Cuyahoga River

    USGS Publications Warehouse

    Brady, Amie M.G.

    2007-01-01

    This study is a continuation of a previous U.S. Geological Survey (USGS) project in cooperation with the National Park Service at Cuyahoga Valley National Park in Brecksville, Ohio. A rapid (1-hour) method for detecting Escherichia coli (E. coli) in water was tested and compared to the standard (24-hour) method for determining E. coli concentrations. Environmental data were collected to determine turbidity, rainfall, and streamflow at the time of sampling. In the previous study (2004-5), data collected were used to develop predictive models to determine recreational water quality in the river at two sites within the park. Data collected during this continued study (2006) were used to test these models. At Jaite, a centrally located site within the park, the model correctly predicted exceedances or nonexceedances of the Ohio Environmental Protection Agency maximum for recreational water quality in 80 percent of samples. At Old Portage, a site near the upstream boundary of the park, the model correctly predicted recreational water quality in 58 percent of samples. All of the data collected in 2004-6 will be used to develop more accurate models for use in future studies. Analysis and discussion of model results are scheduled to be included in an upcoming USGS Scientific Investigations Report.

  20. Identification of Diarrheagenic Escherichia coli Strains from Avian Organic Fertilizers

    PubMed Central

    Puño-Sarmiento, Juan; Gazal, Luis Eduardo; Medeiros, Leonardo P.; Nishio, Erick K.; Kobayashi, Renata K. T.; Nakazato, Gerson

    2014-01-01

    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. PMID:25170683

  1. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli.

    PubMed Central

    Lin, J; Smith, M P; Chapin, K C; Baik, H S; Bennett, G N; Foster, J W

    1996-01-01

    Enterohemorrhagic strains of Escherichia coli must pass through the acidic gastric barrier to cause gastrointestinal disease. Taking into account the apparent low infectious dose of enterohemorrhagic E. coli, 11 O157:H7 strains and 4 commensal strains of E. coli were tested for their abilities to survive extreme acid exposures (pH 3). Three previously characterized acid resistance systems were tested. These included an acid-induced oxidative system, an acid-induced arginine-dependent system, and a glutamate-dependent system. When challenged at pH 2.0, the arginine-dependent system provided more protection in the EHEC strains than in commensal strains. However, the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. Because E. coli must also endure acid stress imposed by the presence of weak acids in intestinal contents at a pH less acidic than that of the stomach, the ability of specific acid resistance systems to protect against weak acids was examined. The arginine- and glutamate-dependent systems were both effective in protecting E. coli against the bactericidal effects of a variety of weak acids. The acids tested include benzoic acid (20 mM; pH 4.0) and a volatile fatty acid cocktail composed of acetic, propionic, and butyric acids at levels approximating those present in the intestine. The oxidative system was much less effective. Several genetic aspects of E. coli acid resistance were also characterized. The alternate sigma factor RpoS was shown to be required for oxidative acid resistance but was only partially involved with the arginine- and glutamate-dependent acid resistance systems. The arginine decarboxylase system (including adi and its regulators cysB and adiY) was responsible for arginine-dependent acid resistance. The results suggest that several acid resistance systems potentially contribute to the survival of pathogenic E. coli in the different acid stress environments of

  2. Developing an extended genomic engineering approach based on recombineering to knock-in heterologous genes to Escherichia coli genome.

    PubMed

    Sukhija, Karan; Pyne, Michael; Ali, Saad; Orr, Valerie; Abedi, Daryoush; Moo-Young, Murray; Chou, C Perry

    2012-06-01

    Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase-flippase recognition targets (FLP-FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P( trc ) and P( araB )), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-D: -1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to "tailor" plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering. PMID:21826554

  3. 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…

  4. Diet, fecal microbiome and Escherichia coli O157:H7 shedding in beef Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga-toxigenic Escherichia coli, such as E. coli O157:H7, are foodborne zoonotic pathogens that can cause severe illness and death in humans. The gastrointestinal tract of ruminant animals has been identified as a primary habitat for E. coli O157:H7, and in cattle the terminal gastrointestinal tra...

  5. 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...

  6. 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...

  7. Escherichia coli O157:H7, diet, and fecal microbiome in beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga-toxigenic Escherichia coli, such as E. coli O157:H7, are foodborne zoonotic pathogens that can cause severe illness and death in humans. The gastrointestinal tract of ruminant animals has been identified as a primary habitat for E. coli O157:H7, and in cattle the terminal gastrointestinal tra...

  8. 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.

  9. 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...

  10. Dietary interactions and interventions affecting Escherichia coli 0157 colonization and shedding in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157 is an important foodborne pathogen affecting human health and the beef cattle industry. Contamination of carcasses at slaughter is correlated to the prevalence of E. coli O157 in cattle feces. Many associations have been made between dietary factors and E. coli O157 prevalenc...

  11. Diet, Escherichia coli O157:H7, and cattle: A review after 10 years

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli are commensal bacteria that can account for up to 1% of the bacterial population of the gut. Ruminant animals are reservoirs of the pathogenic bacteria E. coli O157:H7, and approximately 30% of feedlot cattle shed E. coli O157:H7. Feedlot and high-producing dairy cattle are fed hi...

  12. Diet, Escherichia coli 0157:H7, and cattle, a review after 10 years

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli are commensal bacteria that can account for up to 1% of the bacterial population of the gut. Ruminant animals are reservoirs of the pathogenic bacteria E. coli strain O157:H7 and approximately 30% of feedlot cattle shed E. coli O157:H7. Feedlot and high-producing dairy cattle are ...

  13. 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...

  14. 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...

  15. 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...

  16. Persistence of Escherichia coli O157 and non-O157 strains in agricultural soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin producing Escherichia coli O157 and non-O157 serogroups are known to cause serious diseases in human. However, research on the persistence of E. coli non-O157 serogroups in preharvest environment is limited. In the current study, we compared the survival behavior of E. coli O157 to that ...

  17. 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...

  18. Probiotic Escherichia coli Nissle 1917 reduces growth, Shiga toxin expression, release and thus cytotoxicity of enterohemorrhagic Escherichia coli.

    PubMed

    Mohsin, Mashkoor; Guenther, Sebastian; Schierack, Peter; Tedin, Karsten; Wieler, Lothar H

    2015-01-01

    Due to increased release or production of Shiga toxin by Enterohemorrhagic Escherichia coli (EHEC) after exposure to antimicrobial agents, the role of antimicrobial agents in EHEC mediated infections remains controversial. Probiotics are therefore rapidly gaining interest as an alternate therapeutic option. The well-known probiotic strain Escherichia coli Nissle 1917 (EcN) was tested in vitro to determine its probiotic effects on growth, Shiga toxin (Stx) gene expression, Stx amount and associated cytotoxicity on the most important EHEC strains of serotype O104:H4 and O157:H7. Following co-culture of EcN:EHEC in broth for 4 and 24 h, the probiotic effects on EHEC growth, toxin gene expression, Stx amount and cytotoxicity were determined using quantitative real time-PCR, Stx-ELISA and Vero cytotoxicity assays. Probiotic EcN strongly reduced EHEC numbers (cfu) of O104:H4 up to (68%) and O157:H7 to (72.2%) (p<0.05) in LB broth medium whereas the non-probiotic E. coli strain MG1655 had no effect on EHEC growth. The level of stx expression was significantly down-regulated, particularly for the stx2a gene. The stx down-regulation in EcN co-culture was not due to reduced numbers of EHEC. A significant inhibition in Stx amounts and cytotoxicity were also observed in sterile supernatants of EcN:EHEC co-cultures. These findings indicate that probiotic EcN displays strong inhibitory effects on growth, Shiga toxin gene expression, amount and cytotoxicity of EHEC strains. Thus, EcN may be considered as a putative therapeutic candidate, in particular against EHEC O104:H4 and O157:H7. PMID:25465158

  19. Biosynthesis of two quercetin O-diglycosides in Escherichia coli.

    PubMed

    An, Dae Gyun; Yang, So Mi; Kim, Bong Gyu; Ahn, Joong-Hoon

    2016-06-01

    Various flavonoid glycosides are found in nature, and their biological activities are as variable as their number. In some cases, the sugar moiety attached to the flavonoid modulates its biological activities. Flavonoid glycones are not easily synthesized chemically. Therefore, in this study, we attempted to synthesize quercetin 3-O-glucosyl (1→2) xyloside and quercetin 3-O-glucosyl (1→6) rhamnoside (also called rutin) using two uridine diphosphate-dependent glycosyltransferases (UGTs) in Escherichia coli. To synthesize quercetin 3-O-glucosyl (1→2) xyloside, sequential glycosylation was carried out by regulating the expression time of the two UGTs. AtUGT78D2 was subcloned into a vector controlled by a Tac promoter without a lacI operator, while AtUGT79B1 was subcloned into a vector controlled by a T7 promoter. UDP-xyloside was supplied by concomitantly expressing UDP-glucose dehydrogenase (ugd) and UDP-xyloside synthase (UXS) in the E. coli. Using these strategies, 65.0 mg/L of quercetin 3-O-glucosyl (1→2) xyloside was produced. For the synthesis of rutin, one UGT (BcGT1) was integrated into the E. coli chromosome and the other UGT (Fg2) was expressed in a plasmid along with RHM2 (rhamnose synthase gene 2). After optimization of the initial cell concentration and incubation temperature, 119.8 mg/L of rutin was produced. The strategies used in this study thus show promise for the synthesis of flavonoid diglucosides in E. coli. PMID:26931782

  20. Escherichia coli and the Emergence of Molecular Biology.

    PubMed

    Ullmann, Agnes

    2011-12-01

    The creation of the "Phage group" by M. Delbrück, S. E. Luria, and A. D. Hershey in 1940 at Cold Spring Harbor played a crucial role in the development of molecular biology. In the 1940s, working with Escherichia coli and its viruses, Luria and Delbrück discovered the spontaneous nature of bacterial mutations and Hershey described recombination in bacteriophages and demonstrated with M. Chase that the genetic material that infects bacteria is DNA. At the same time, S. Benzer defined the structure of a functional genetic unit and J. Lederberg and E. Tatum discovered sexual recombination between bacteria. Some years later, Lederberg's group discovered extrachromosomal particles, the plasmids, and a novel way of genetic transfer through bacteriophages, called transduction. In 1949, at the Pasteur Institute in Paris, A. Lwoff uncovered the mechanism of lysogeny. Shortly afterwards, F. Jacob and E. Wollman unraveled the mechanism of the sexual process in E. coli and established the circularity of the bacterial chromosome. In the 1960s, J. Monod and F. Jacob, by genetic analysis of the E. coli lactose system, proposed the operon model for gene regulation and introduced the concept of messenger RNA. The elucidation of the double helix structure of DNA in 1953 by F. Crick and J. Watson had major consequences: the establishment of the copying mechanism (Meselson and Stahl), the discovery of the nature of the genetic code (S. Brenner) leading to its deciphering. E. coli and its phages were instrumental in the development of recombinant DNA technology based on the discovery of the restriction-modification system by W. Arber. PMID:26442505

  1. 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. PMID:25411099

  2. Engineering an Escherichia coli platform to synthesize designer biodiesels.

    PubMed

    Wierzbicki, Michael; Niraula, Narayan; Yarrabothula, Akshitha; Layton, Donovan S; Trinh, Cong T

    2016-04-20

    Biodiesels, fatty acid esters (FAEs), can be synthesized by condensation of fatty acid acyl CoAs and alcohols via a wax ester synthase in living cells. Biodiesels have advantageous characteristics over petrodiesels such as biodegradability, a higher flash point, and less emission. Controlling fatty acid and alcohol moieties are critical to produce designer biodiesels with desirable physiochemical properties (e.g., high cetane number, low kinematic viscosity, high oxidative stability, and low cloud point). Here, we developed a flexible framework to engineer Escherichia coli cell factories to synthesize designer biodiesels directly from fermentable sugars. In this framework, we designed each FAE pathway as a biodiesel exchangeable production module consisting of acyl CoA, alcohol, and wax ester synthase submodules. By inserting the FAE modules in an engineered E. coli modular chassis cell, we generated E. coli cell factories to produce targeted biodiesels (e.g., fatty acid ethyl (FAEE) and isobutyl (FAIbE) esters) with tunable and controllable short-chain alcohol moieties. The engineered E. coli chassis carrying the FAIbE production module produced 54mg/L FAIbEs with high specificity, accounting for>90% of the total synthesized FAEs and ∼4.7 fold increase in FAIbE production compared to the wildtype. Fed-batch cultures further improved FAIbE production up to 165mg/L. By mixing ethanol and isobutanol submodules, we demonstrated controllable production of mixed FAEEs and FAIbEs. We envision the developed framework offers a flexible, alternative route to engineer designer biodiesels with tunable and controllable properties using biomass-derived fermentable sugars. PMID:26953744

  3. Source and regulation of flux variability in Escherichia coli

    PubMed Central

    2014-01-01

    Background Metabolic responses are essential for the adaptation of microorganisms to changing environmental conditions. The repertoire of flux responses that the metabolic network can display in different external conditions may be quantified applying flux variability analysis to genome-scale metabolic reconstructions. Results A procedure is developed to classify and quantify the sources of flux variability. We apply the procedure to the latest Escherichia coli metabolic reconstruction, in glucose minimal medium, with an additional constraint to account for the mechanism coordinating carbon and nitrogen utilization mediated by α-ketoglutarate. Flux variability can be decomposed into three components: internal, external and growth variability. Unexpectedly, growth variability is the only significant component of flux variability in the physiological ranges of glucose, oxygen and ammonia uptake rates. To obtain substantial increases in metabolic flexibility, E. coli must decrease growth rate to suboptimal values. This growth-flexibility trade-off gives a straightforward interpretation to recent work showing that most overall cell-to-cell flux variability in a population of E. coli can be attained sampling a small number of enzymes most likely to constrain cell growth. Importantly, it provides an explanation for the global reorganization occurring in metabolic networks during adaptations to environmental challenges. The calculations were repeated with a pathogenic strain and an old reconstruction of the commensal strain, having less than 50% of the reactions of the latest reconstruction, obtaining the same general conclusions. Conclusions In E. coli growing on glucose, growth variability is the only significant component of flux variability for all physiological conditions explored. Increasing flux variability requires reducing growth to suboptimal values. The growth-flexibility trade-off operates in physiological and evolutionary adaptations, and provides an

  4. Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli isolates(72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that in nonhuman hosts E. coli O157:H7 strains function ...

  5. Atypical biogroups of Escherichia coli found in clinical specimens and description of Escherichia hermannii sp. nov.

    PubMed

    Brenner, D J; Davis, B R; Steigerwalt, A G; Riddle, C F; McWhorter, A C; Allen, S D; Farmer, J J; Saitoh, Y; Fanning, G R

    1982-04-01

    DNA relatedness was used to define the biochemical boundaries of Escherichia coli. A large number of biochemically atypical strains were shown to belong to biogroups of E. coli. These included strains negative in reactions for indole, all three decarboxylases, D-mannitol, lactose, or methyl red and strains positive in reactions for H2S, urea, citrate, KCN, adonitol, myo-inositol, or phenylalanine deaminase. Frequency and source data are presented for these atypical E. coli biogroups. One group of KCN-positive, cellobiose-positive, yellow-pigmented strains was 84 to 91% interrelated but only 35 to 45% related to E. coli. The name Escherichia hermannii sp. nov. is proposed for this group of organisms that was formerly called Enteric Group 11 by the Enteric Section, Centers for Disease Control, Atlanta, GA. Twenty-nine strains of E. hermannii have been isolated in the United States from a variety of clinical sources, principally wounds, sputum, and stools. Three additional strains were isolated from food. E. hermannii strains are gram-negative, oxidase-negative, fermentative, motile rods. In addition to yellow pigment and positive KCN and cellobiose tests, the biochemical reactions characteristic of 32 strains of E. hermannii were as follows: gas from D-glucose, acid from D-glucose, maltose, D-xylose, L-arabinose, L-rhamnose, and D-mannitol; no acid from adonitol or inositol; variable acid production from lactose and sucrose; positive tests for indole, methyl red, and mucate; negative tests for Voges-Proskauer. Simmons citrate, H2S, urea, phenylalanine deaminase, and gelatin hydrolysis; negative or delayed test for L-lysine decarboxylase and negative test for L-arginine dihydrolase; and positive test for ornithine decarboxylase. E. hermannii strains were resistant to penicillin, ampicillin, and carbenicillin and sensitive to other commonly used antibiotics. Wounds account for almost 50% of human isolates of E. hermannii, followed by sputum or lung isolates (ca. 25

  6. 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

  7. 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. PMID

  8. Improved genetically modified Escherichia coli strain for prescreening antineoplastic agents.

    PubMed Central

    Bartus, H R; Mirabelli, C K; Auerbach, J I; Shatzman, A R; Taylor, D P; Johnson, R K; Rosenberg, M; Crooke, S T

    1984-01-01

    Clinical experience suggests that drugs that interact with and damage DNA are useful in cancer chemotherapy (H. Umezawa , p. 43-72, in V. T. DeVita , Jr., and H. Busch [ed.], Methods in Cancer Research; Cancer Drug Development, vol. XVI, 1979). Prescreening systems for antitumor agents in natural products require assays that are exquisitely sensitive, since the active components are often produced in quantities of micrograms per milliliter or less. One assay used to identify agents that interact with DNA is the biochemical induction assay, utilizing Escherichia coli BR 513 (R. K. Elespuru and R. J. White, Cancer Res. 43:2819-2830, 1983). In this paper we describe a genetic modification of strain BR 513 that displays an expanded spectrum of activity. This strain may provide an improved prescreen for detecting natural products that interact with DNA. PMID:6203484

  9. SOS Response Induces Persistence to Fluoroquinolones in Escherichia coli

    PubMed Central

    Dörr, Tobias; Lewis, Kim; Vulić, Marin

    2009-01-01

    Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance. We investigated persistence to the fluoroquinolone ciprofloxacin in Escherichia coli. Our data show that a majority of persisters to ciprofloxacin were formed upon exposure to the antibiotic, in a manner dependent on the SOS gene network. These findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means. SOS-induced persistence is a novel mechanism by which cells can counteract DNA damage and promote survival to fluoroquinolones. This unique survival mechanism may be an important factor influencing the outcome of antibiotic therapy in vivo. PMID:20011100

  10. Kinetics of minichromosome replication in Escherichia coli B/r.

    PubMed

    Leonard, A C; Hucul, J A; Helmstetter, C E

    1982-02-01

    Replication control of the minichromosome pAL2 was found to differ from that of the chromosome in synchronously dividing populations of Escherichia coli B/r. Initiation of minichromosome replication took place at an increasing rate throughout synchronous growth. No coupling to initiation of chromosome replication was detected. Minichromosome replication was further examined in a dnaA5(Ts) temperature-sensitive initiation mutant. When cultures held at nonpermissive temperature (41 degrees C) for 60 min were shifted to permissive temperature (25 degrees C), initiation of both pAL2 and chromosome replication ensued in two waves spaced 25 to 35 min apart. Evidence is presented that minichromosomes terminate replication by passing slowly through a series of dimeric intermediate forms before reaching the closed circular monomeric form. The consequence of this slow passage as a rate-limiting step in the initiation reaction is discussed. PMID:7035432

  11. Coordinate initiation of chromosome and minichromosome replication in Escherichia coli.

    PubMed Central

    Helmstetter, C E; Leonard, A C

    1987-01-01

    Escherichia coli minichromosomes harboring as little as 327 base pairs of DNA from the chromosomal origin of replication (oriC) were found to replicate in a discrete burst during the division cycle of cells growing with generation times between 25 and 60 min at 37 degrees C. The mean cell age at minichromosome replication coincided with the mean age at initiation of chromosome replication at all growth rates, and furthermore, the age distributions of the two events were indistinguishable. It is concluded that initiation of replication from oriC is controlled in the same manner on minichromosomes and chromosomes over the entire range of growth rates and that the timing mechanism acts within the minimal oriC nucleotide sequence required for replication. Images PMID:3301802

  12. Kinetics of minichromosome replication in Escherichia coli B/r.

    PubMed Central

    Leonard, A C; Hucul, J A; Helmstetter, C E

    1982-01-01

    Replication control of the minichromosome pAL2 was found to differ from that of the chromosome in synchronously dividing populations of Escherichia coli B/r. Initiation of minichromosome replication took place at an increasing rate throughout synchronous growth. No coupling to initiation of chromosome replication was detected. Minichromosome replication was further examined in a dnaA5(Ts) temperature-sensitive initiation mutant. When cultures held at nonpermissive temperature (41 degrees C) for 60 min were shifted to permissive temperature (25 degrees C), initiation of both pAL2 and chromosome replication ensued in two waves spaced 25 to 35 min apart. Evidence is presented that minichromosomes terminate replication by passing slowly through a series of dimeric intermediate forms before reaching the closed circular monomeric form. The consequence of this slow passage as a rate-limiting step in the initiation reaction is discussed. PMID:7035432

  13. Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase.

    PubMed

    Wang, Dezheng; Wang, Cheng; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-01-01

    Glutathione (GSH) is an important bioactive substance applied widely in pharmaceutical and food industries. Due to the strong product inhibition in the GSH biosynthetic pathway, high levels of intracellular content, yield and productivity of GSH are difficult to achieve. Recently, a novel bifunctional GSH synthetase was identified to be less sensitive to GSH. A recombinant Escherichia coli strain expressing gshF encoding the bifunctional glutathione synthetase of Streptococcus thermophilus was constructed for GSH production. In this study, efficient GSH production using this engineered strain was investigated. The cultivation process was optimized by controlling dissolved oxygen (DO), amino acid addition and glucose feeding. 36.8 mM (11.3 g/L) GSH were formed at a productivity of 2.06 mM/h when the amino acid precursors (75 mM each) were added and glucose was supplied as the sole carbon and energy source. PMID:26586402

  14. Selection of quiescent Escherichia coli with high metabolic activity.

    PubMed

    Sonderegger, Marco; Schümperli, Michael; Sauer, Uwe

    2005-01-01

    Sustained metabolic activity in non-growing, quiescent cells can increase the operational life-span of bio-processes and improve process economics by decoupling production from cell growth. Because of the ill-defined molecular nature of this phenotype, we developed selection protocols for the evolution of quiescent Escherichia coli mutants that exhibit high metabolic activity in ammonium starvation-induced stationary phase. The best enrichment procedures were continuously or discontinuously fed ammonium-limited chemostat cultures with a very low dilution rate of 0.03 h(-1). After 40 generations of selection, improved mutants with up to doubled catabolic rates in stationary phase were isolated. The metabolically most active clones were identified by screening for high specific glucose uptake rates during ammonium starvation-induced stationary phase in deep-well microtiter plates. PMID:15721805

  15. Expanding the genetic code of Escherichia coli with phosphotyrosine.

    PubMed

    Fan, Chenguang; Ip, Kevan; Söll, Dieter

    2016-09-01

    Protein phosphorylation is one of the most important post-translational modifications in nature. However, the site-specific incorporation of O-phosphotyrosine into proteins in vivo has not yet been reported. Endogenous phosphatases present in cells can dephosphorylate phosphotyrosine as a free amino acid or as a protein residue. Therefore, we deleted the genes of five phosphatases from the genome of Escherichia coli with the aim of stabilizing phosphotyrosine. Together with an engineered aminoacyl-tRNA synthetase (derived from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase) and an elongation factor Tu variant, we were able to cotranslationally incorporate O-phosphotyrosine into the superfolder green fluorescent protein at a desired position in vivo. This system will facilitate future studies of tyrosine phosphorylation. PMID:27477338

  16. 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). PMID:24742948

  17. Implications of enterotoxigenic Escherichia coli genomics for vaccine development.

    PubMed

    Sjöling, Åsa; von Mentzer, Astrid; Svennerholm, Ann-Mari

    2015-04-01

    Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality caused by diarrhea in children less than 5 years of age in low- and middle-income countries. Despite a wealth of research elucidating the mechanisms of disease, the immunological responses and vaccine development, ETEC is still relatively uncharacterized when it comes to regulation of virulence and detailed immune mechanisms. The recent emergence of next-generation sequencing now offers the possibility to screen genomes of ETEC strains isolated globally to identify novel vaccine targets in addition to those already established. In this review, we discuss how recent findings on ETEC genomics using novel sequencing techniques will aid in finding novel protective antigens that can be used in vaccine approaches. PMID:25540974

  18. Chromosome segregation control by Escherichia coli ObgE GTPase.

    PubMed

    Foti, James J; Persky, Nicole S; Ferullo, Daniel J; Lovett, Susan T

    2007-07-01

    Escherichia coli cells depleted of the conserved GTPase, ObgE, show early chromosome-partitioning defects and accumulate replicated chromosomes in which the terminus regions are colocalized. Cells lacking ObgE continue to initiate replication, with a normal ratio of the origin to terminus. Localization of the SeqA DNA binding protein, normally seen as punctate foci, however, was disturbed. Depletion of ObgE also results in cell filamentation, with polyploid DNA content. Depletion of ObgE did not cause lethality, and cells recovered fully after expression of ObgE was restored. We propose a model in which ObgE is required to license chromosome segregation and subsequent cell cycle events. PMID:17578452

  19. Adaptive Reversion of a Frameshift Mutation in Escherichia Coli

    PubMed Central

    Cairns, J.; Foster, P. L.

    1991-01-01

    Mutation rates are generally thought not to be influenced by selective forces. This doctrine rests on the results of certain classical studies of the mutations that make bacteria resistant to phages and antibiotics. We have studied a strain of Escherichia coli which constitutively expresses a lacI-lacZ fusion containing a frameshift mutation that renders it Lac(-). Reversion to Lac(+) is a rare event during exponential growth but occurs in stationary cultures when lactose is the only source of energy. No revertants accumulate in the absence of lactose, or in the presence of lactose if there is another, unfulfilled requirement for growth. The mechanism for such mutation in stationary phase is not known, but it requires some function of RecA which is apparently not required for mutation during exponential growth. PMID:1916241

  20. Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

    SciTech Connect

    Roberston, E.F.; Hoyt, J.C.; Reeves, H.C.

    1987-05-01

    Escherichia coli isocitrate lyase can be phosphorylated in vitro in an ATP-dependent reaction. Partially purified extracts were incubated with ..gamma..-/sup 32/P-ATP and analyzed by two-dimensional polyacrylamide gel electrophoresis followed by a Western blot and autoradiography. Radioactivity was associated with the lyase only when blotting was performed under alkaline conditions. This suggests that phosphate groups are attached to the lyase via an acid-labile P-N bond rather than a more stable P-O bond. Treatment of the lyase with diethyl pyrocarbonate, a histidine modifying agent, blocks incorporation of /sup 32/P-phosphate. Treatment with phosphoramidate, a histidine phosphorylating agent, alters the isoelectric point of the lyase suggesting that the enzyme can be phosphorylated at histidine residues. Loss of catalytic activity after treatment with potato acid phosphatase indicates that isocitrate lyase activity may be modulated by phosphorylation.

  1. A series of template plasmids for Escherichia coli genome engineering.

    PubMed

    Deb, Shalini S; Reshamwala, Shamlan M S; Lali, Arvind M

    2016-06-01

    Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated. PMID:27071533

  2. Escherichia coli produces linoleic acid during late stationary phase.

    PubMed Central

    Rabinowitch, H D; Sklan, D; Chace, D H; Stevens, R D; Fridovich, I

    1993-01-01

    Escherichia coli produces linoleic acid in the late stationary phase. This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium. The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography. The linoleic acid methyl ester was identified by its mass spectrum. Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator. In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells. PMID:8366020

  3. Intramolecular dynamics of structure of alkaline phosphatase from Escherichia coli

    NASA Astrophysics Data System (ADS)

    Mazhul, Vladimir M.; Mjakinnik, Igor V.; Volkova, Alena N.

    1995-01-01

    The luminescent analysis with nano- and millisecond time resolution of intramolecular dynamics of Escherichia coli alkaline phosphatase was carried out. The effect of pH within the range 7.2 - 9.0, thermal inactivation, limited proteolysis by trypsin, binding of pyrophosphate, interconversion of enzyme and apoenzyme, the replacement of Zn2+ and Mg2+ in the active site by Cd2+ and Ni2+ on the spectral and kinetic parameters of luminescence was investigated. The essential changes of the level of nano- and millisecond dynamics of protein structure were found to correlate with the shift of enzymatic activity. The importance of small- and large-scale flexibility of protein structure for the act of enzymatic catalysis realization was shown.

  4. Uropathogenic Escherichia coli Epigenetically Manipulate Host Cell Death Pathways.

    PubMed

    Zhang, Zhengguo; Wang, Ming; Eisel, Florian; Tchatalbachev, Svetlin; Chakraborty, Trinad; Meinhardt, Andreas; Bhushan, Sudhanshu

    2016-04-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in human. It is well established that UPEC can subvert innate immune responses, but the role of UPEC in interfering with host cell death pathways is not known. Here, we show that UPEC abrogates activation of the host cell prosurvival protein kinase B signaling pathway, which results in the activation of mammalian forkhead box O (FOXO) transcription factors. Although FOXOs were localized in the nucleus and showed increased DNA-binding activity, no change in the expression levels of FOXO target genes were observed. UPEC can suppress BIM expression induced by LY249002, which results in attenuation of caspase 3 activation and blockage of apoptosis. Mechanistically, BIM expression appears to be epigenetically silenced by a decrease in histone 4 acetylation at the BIM promoter site. Taken together, these results suggest that UPEC can epigenetically silence BIM expression, a molecular switch that prevents apoptosis. PMID:26621912

  5. Structure of the Cyclomodulin Cif from Pathogenic Escherichia coli

    SciTech Connect

    Hsu, Y.; Jubelin, G; Taieb, F; Nougayrède, J; Oswald, E; Stebbins, C

    2008-01-01

    Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.

  6. Regulation of Phospholipid Synthesis in Escherichia coli by Guanosine Tetraphosphate

    PubMed Central

    Merlie, John P.; Pizer, Lewis I.

    1973-01-01

    Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with 32P-inorganic phosphate and 32P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells. Images PMID:4583220

  7. Parallel Mapping of Antibiotic Resistance Alleles in Escherichia coli

    PubMed Central

    Mortazavi, Pooneh; Knight, Rob; Gill, Ryan T.

    2016-01-01

    Chemical genomics expands our understanding of microbial tolerance to inhibitory chemicals, but its scope is often limited by the throughput of genome-scale library construction and genotype-phenotype mapping. Here we report a method for rapid, parallel, and deep characterization of the response to antibiotics in Escherichia coli using a barcoded genome-scale library, next-generation sequencing, and streamlined bioinformatics software. The method provides quantitative growth data (over 200,000 measurements) and identifies contributing antimicrobial resistance and susceptibility alleles. Using multivariate analysis, we also find that subtle differences in the population responses resonate across multiple levels of functional hierarchy. Finally, we use machine learning to identify a unique allelic and proteomic fingerprint for each antibiotic. The method can be broadly applied to tolerance for any chemical from toxic metabolites to next-generation biofuels and antibiotics. PMID:26771672

  8. Peptidoglycan biosynthesis in stationary-phase cells of Escherichia coli.

    PubMed Central

    Blasco, B; Pisabarro, A G; de Pedro, M A

    1988-01-01

    The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursors into macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to the insertion of 1.3 X 10(4) disaccharide units min-1 cell-1. Afterwards, the rate of incorporation dropped drastically (90%) to a low but still detectable level. Incorporation during stationary phase did not result in an increased amount of total murein in the culture, suggesting that it was related to a turnover process. Analysis of the effects of a number of beta-lactam antibiotics indicated that incorporation of murein precursors in stationary-phase cells was mediated by penicillin-binding proteins, suggesting that the activity of penicillin-binding protein 2 was particularly relevant to this process. PMID:3141382

  9. 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

  10. Escherichia coli gene that controls sensitivity to alkylating agents.

    PubMed Central

    Yamamoto, Y; Katsuki, M; Sekiguchi, M; Otsuji, N

    1978-01-01

    A new type of Escherichia coli mutant which shows increased sensitivity to methyl methane sulfonate but not to UV light or to gamma rays was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant is unable to reactivate phage lambdavir or double-stranded phiX174 DNA (replicative form) that had been treated with methyl methane sulfonate. The mutant is sensitive to other alkylating agents, such as ethyl methane sulfonate, mitomycin C, and N-methyl-N'-nitro-N-nitrosoguanidine, as well. It grows normally and exhibits almost normal recombination proficiency. The mutant possesses normal levels of DNA polymerase I, exonuclease I, exonuclease V, endonuclease specific for methyl methane sulfonate-treated DNA, and 3-methyladenine-DNA glycosidase activities. The genetic locus responsible has been named alk and is located near his on the chromosome. PMID:353028

  11. Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli.

    PubMed

    Ge, Baosheng; Sun, Haixiang; Feng, Yang; Yang, Jinying; Qin, Song

    2009-03-01

    Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g l(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein. PMID:19269586

  12. 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. PMID:26804288

  13. 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

  14. Lateral diffusion of proteins in the periplasm of Escherichia coli.

    PubMed Central

    Brass, J M; Higgins, C F; Foley, M; Rugman, P A; Birmingham, J; Garland, P B

    1986-01-01

    We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis. Images PMID:3005237

  15. Effect of temperature on motility and chemotaxis of Escherichia coli.

    PubMed Central

    Maeda, K; Imae, Y; Shioi, J I; Oosawa, F

    1976-01-01

    The swimming velocity of Escherichia coli at various constant temperatures was found to increase with increasing temperature. The frequency of tumbling had a peak at 34 degrees C and was very low both at 20 and at 39 degrees C. The swimming tracks near the surface of a slide glass showed curves, and the curvature increased the temperature. When the temperature of a bacterial suspension was suddenly changed, a transient change of the tumbling frequency was observed. A temperature drop induced a temporary increase in the tumbling frequency, and a quick rise of temperature, on the other hand, resulted in a temporary suppression of the tumbling. These dynamic responses to sudden changes of temperature was not observed in the smoothly swimming nonchemotactic strains bearing the mutations cheA and cheC and also in a mutant with the metF mutation under a smooth swimming condition. Images PMID:783127

  16. Modeling and observer design for recombinant Escherichia coli strain.

    PubMed

    Nadri, M; Trezzani, I; Hammouri, H; Dhurjati, P; Longin, R; Lieto, J

    2006-03-01

    A mathematical model for recombinant bacteria which includes foreign protein production is developed. The experimental system consists of an Escherichia Coli strain and plasmid pIT34 containing genes for bioluminescence and production of a protein, beta-galactosidase. This recombinant strain is constructed to facilitate on-line estimation and control in a complex bioprocess. Several batch experiments are designed and performed to validate the developed model. The design of a model structure, the identification of the model parameters and the estimation problem are three parts of a joint design problem. A nonlinear observer is designed and an experimental evaluation is performed on a batch fermentation process to estimate the substrate consumption. PMID:16411071

  17. Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

    PubMed

    Gholizadeh, A; Kohnehrouz, B Baghban; Santha, I M; Lodha, M L; Kapoor, H C

    2005-09-01

    A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714. PMID:16266271

  18. Interfering with Different Steps of Protein Synthesis Explored by Transcriptional Profiling of Escherichia coli K-12†

    PubMed Central

    Sabina, Jeffrey; Dover, Nir; Templeton, Lori J.; Smulski, Dana R.; Söll, Dieter; LaRossa, Robert A.

    2003-01-01

    Escherichia coli responses to four inhibitors that interfere with translation were monitored at the transcriptional level. A DNA microarray method provided a comprehensive view of changes in mRNA levels after exposure to these agents. Real-time reverse transcriptase PCRanalysis served to verify observations made with microarrays, and a chromosomal grpE::lux operon fusion was employed to specifically monitor the heat shock response. 4-Azaleucine, a competitive inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock response. Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase activity, resulted in changes reminiscent of the stringent response. Treatment with kasugamycin and puromycin (targeting ribosomal subunit association as well as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal protein operons. Abundant biosynthetic transcripts were often significantly diminished after treatment with any of these agents. Exposure of a relA strain to mupirocin resulted in accumulation of ribosomal protein operon transcripts. However, the relA strain's response to the other inhibitors was quite similar to that of the wild-type strain. PMID:14526028

  19. The MazF-regulon: a toolbox for the post-transcriptional stress response in Escherichia coli.

    PubMed

    Sauert, Martina; Wolfinger, Michael T; Vesper, Oliver; Müller, Christian; Byrgazov, Konstantin; Moll, Isabella

    2016-08-19

    Flexible adaptation to environmental stress is vital for bacteria. An energy-efficient post-transcriptional stress response mechanism in Escherichia coli is governed by the toxin MazF. After stress-induced activation the endoribonuclease MazF processes a distinct subset of transcripts as well as the 16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes' are specific for the MazF-processed mRNAs, the translational program is changed. To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation coupled with RNA-seq analysis) and analyzed alterations introduced into the transcriptome and translatome after mazF overexpression. Unexpectedly, our results reveal that the corresponding protein products are involved in all cellular processes and do not particularly contribute to the general stress response. Moreover, our findings suggest that translational reprogramming serves as a fast-track reaction to harsh stress and highlight the so far underestimated significance of selective translation as a global regulatory mechanism in gene expression. Considering the reported implication of toxin-antitoxin (TA) systems in persistence, our results indicate that MazF acts as a prime effector during harsh stress that potentially introduces translational heterogeneity within a bacterial population thereby stimulating persister cell formation. PMID:26908653

  20. Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET.

    PubMed

    Buskiewicz, Iwona; Peske, Frank; Wieden, Hans-Joachim; Gryczynski, Ignacy; Rodnina, Marina V; Wintermeyer, Wolfgang

    2005-08-12

    The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure of Ffh alone and in the complex with 4.5S RNA by measuring distances between different positions within Ffh and between Ffh and 4.5S RNA by fluorescence resonance energy transfer (FRET). According to the FRET distances, NG and M domains in free Ffh are in close contact, as in the A/A arrangement in the crystal structure of Ffh from Thermus aquaticus, in agreement with the formation of a crosslink between cysteine residues at two critical positions in the G and M domains. Upon Ffh binding to 4.5S RNA or a 61 nucleotide fragment comprising internal loops A-C, the G and M domains move apart to assume a more open conformation, as indicated by changes of FRET distances. The movement is smaller when Ffh binds to a 49 nucleotide fragment of 4.5S RNA comprising only internal loops A and B, i.e. lacking the binding site of the NG domain. The FRET results suggest that in the SRP complex 4.5S RNA is present in a bent, rather than extended, conformation. The domain rearrangement of Ffh that takes place upon formation of the SRP is probably important for subsequent steps of membrane targeting, including interactions with the translating ribosome and the SRP receptor. PMID:16005894