Escherichia coli

Anal Chem, 2001 Sep 1, 73(17), 4063 - 70
Virtual 2-D gel electrophoresis: visualization and analysis of the E . coli proteome by mass spectrometry; Loo RR et al.; Mass spectrometric surface analysis of isoelectric focusing gels provides an ultrasensitive approach to proteome analysis . This "virtual 2-D gel" approach, in which mass spectrometry is substituted for the size-based separation of SDS-PAGE, provides advantages in mass resolution and accuracy over classical 2-D gels and can be readily automated . Protein identities can be postulated from molecular mass (+/-0.1-0.2% for proteins of <50 kDa in size) and pI (+/-0.3 pH unit) and confirmed by MALDI in-source decay of the intact protein (providing sequence spanning up to 43 residues) or by peptide mass mapping following gel-wide chemical cleavage . Additionally, posttranslational modifications such as fatty acid acylation can be detected by the mass-resolved heterogeneity of component hydrocarbon chains . Sensitivity was evaluated by comparing the number of proteins detected by this method to equivalently loaded silver-stained 2-D gels . In the 5.7-6.0 pH range, E . coli is predicted to contain 435 proteins; virtual 2-D gels found 250 proteins ranging from >2 to <120 kDa in size present at levels to tens of femtomoles, as compared to the 100 proteins found by silver-staining 2-D gels . Extrapolating this result to the total theoretical proteome suggests that this technology is capable of detecting over 2500 E . coli proteins.

Protein Sci, 2001 Oct, 10(10), 2037 - 49
Exploring the conformational equilibrium of E . coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy; van den Berg PA et al.; The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy . The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably . Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S . Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation . The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation . The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation . Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation . Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation . Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model . Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S . High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.

Protein Sci, 2001 Oct, 10(10), 2028 - 36
Examination of the folding of E . coli CspA through tryptophan substitutions; Vu DM et al.; Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an apparent two-state mechanism . However, recent time-resolved infrared (IR) temperature-jump experiments indicate that the folding trajectory of CspA may be more complicated . The sole tryptophan of wild-type CspA (Trp11), which is used to monitor the folding process by fluorescence spectroscopy, is located in an unusual aromatic cluster on the surface of CspA within the nucleic acid binding site . To gain a more global picture of the folding kinetics of CspA and to determine if there are any previously undetected intermediates, we have introduced a second tryptophan at three different surface locations in the protein . The three mutations did not significantly alter the tertiary structure of CspA, although two of the substitutions were found to be slightly stabilizing . Two-state folding, as detected by stopped-flow fluorescence spectroscopy, is preserved in all three mutants . These results indicate that the fast folding of CspA is driven by a concerted mechanism.

Protein Sci, 2001 Oct, 10(10), 1989 - 2001
Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E . coli SSB cross-link to DNA; Steen H et al.; Protein-nucleic acid complexes are commonly studied by photochemical cross-linking . UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site . Mass spectrometry is becoming increasingly popular for characterization of purified peptide-nucleic acid heteroconjugates derived from UV cross-linked protein-nucleic acid complexes . The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates . Sample preparation of the peptide-nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure . This study demonstrates the performance of four different MS-based strategies to characterize E . coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer . Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis . Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis . Detailed characterization of the peptidic component of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues . Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry identified tryptophan-54 and tryptophan-88 as the sites of cross-linking . Although the UV-cross-linking yield of the protein-DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.

Vopr Med Khim, 2001 May-Jun, 47(3), 315 - 28
{Association of cytochrome P450 2B4 with molecular chaperones in heterological expression in E coli.}; Prozorovskii TV et al.; To produce a water-soluble form of microsomal P450 2B4, fusion proteins with glutathione-S-transpherase were genetically engineered . Specific proteolitic sites recognized by Factor Xa and Thrombin have been introduced into N-terminus of P450 2B4 (46-49), lacking signal anchor sequence (2-27) . It was supposed that proteolysis at this site could give the possibility to produce protein lacking hydrophobic N-terminus sequence (1-49) . However, it was shown that given region in P450 2B4 his resistant against specific proteinase action . Positive result has been obtained at specific proteolysis with IgA endoproteinase, recognizing the native sequence PPGP (31-34) in P450 2B4 . Thus, at first time truncated form of cytochrome 2B4, lacking its 33 N-terminal amino acid residues has been created . It was found that the expression of genetically engineered variants of GST-2B4 in Escherichia coli is accompanied by tight complex formation with molecular chaperones GroEL and DnaK . Dissociation of the complex occurred after proteolysis in: linker sequence (position 6-7) between C-terminal part of GST domain and N-terminal part of 2B4, and also before N-terminal methionine 2B4 and at position 33-34 (2B4) . These results suggest the possibility that interaction with a GroEL/DnaK molecular chaperones may be requirement for correct folding of eukaryotic cytochrome 2B4 during its biosynthesis in E . coli.

Prog Nucleic Acid Res Mol Biol, 2001, 68, 349 - 64
Potential double-flipping mechanism by E . coli MutY; House PG et al.; To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted . MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation . However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency . Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A . The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY . Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy . The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase . Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.

Curr Biol, 2001 Sep 4, 11(17), 1369 - 73
Computational identification of noncoding RNAs in E . coli by comparative genomics; Rivas E et al.; Some genes produce noncoding transcripts that function directly as structural, regulatory, or even catalytic RNAs {1, 2} . Unlike protein-coding genes, which can be detected as open reading frames with distinctive statistical biases, noncoding RNA (ncRNA) gene sequences have no obvious inherent statistical biases {3} . Thus, genome sequence analyses reveal novel protein-coding genes, but any novel ncRNA genes remain invisible . Here, we describe a computational comparative genomic screen for ncRNA genes . The key idea is to distinguish conserved RNA secondary structures from a background of other conserved sequences using probabilistic models of expected mutational patterns in pairwise sequence alignments . We report the first whole-genome screen for ncRNA genes done with this method, in which we applied it to the "intergenic" spacers of Escherichia coli using comparative sequence data from four related bacteria . Starting from >23,000 conserved interspecies pairwise alignments, the screen predicted 275 candidate structural RNA loci . A sample of 49 candidate loci was assayed experimentally . At least 11 loci expressed small, apparently noncoding RNA transcripts of unknown function . Our computational approach may be used to discover structural ncRNA genes in any genome for which appropriate comparative genome sequence data are available.

Gravit Space Biol Bull, 1997 Jun, 10(2), 43 - 7
A mechanosensitive channel protein and its gene in E . coli; Blount P et al.; Receptor molecules that respond directly to gravity, touch, vibration, or osmotic pressure are inferred from their functions but not yet characterized as isolated proteins or products of cloned genes . These receptors are often in low abundance and in animal and plant tissues that are inaccessible, thus making biochemical analysis difficult . Yet, the application of the sensitive patch-clamp technique to measure transmembrane currents has demonstrated the ubiquity of ion channels whose opening is favored by membrane stretch forces . We have discovered in E . coli the activity of a mechanosensitive ion channel of large conductance (MscL), and have successfully isolated the corresponding protein and gene (Sukharev et al . 1994a) . MscL channel appears to respond directly to stretch force in the lipid bilayer since it is active in artificial patches having only highly enriched MscL protein and lipids . Structurally, MscL is an integral membrane protein of only 136 amino-acid residues . Each channel pore is likely to be enclosed by six assembled MscL subunits . Hydropathy analysis suggests that the protein is largely hydrophobic with a more hydrophilic carboxyl tail . Targeted deletions and substitutions show that not all regions of the molecule contribute to channel function; however, strategic single amino-acid changes can alter channel kinetics and mechanosensitivity . MscL and its gene now form the first tangible system to study mechanosensing using a combination of genetic, biochemical, and biophysical techniques.

Adv Space Res, 1994 Oct, 14(10), 203 - 6
Heavy ion induced DNA double strand breaks in cells of E . coli; Schafer M et al.; Vegetative cells of E . coli differing in their radiosensitivity have been used in heavy ion irradiation experiment . Besides inactivation measurements also the induction of DNA double strand breaks (DSB) have been measured using the method of pulse-field gel electrophoresis . This method allows to separate linear DNA with length up to 8 Mio base pairs . After irradiation with heavy ions we find a higher amount of low molecular weight fragments when compared to sparsely ionizing radiation . This agrees with the idea that heavy ions as a structured radiation have a high probability to induce more than one strand break in a DNA molecule if the particle hits the DNA . The amount of intact DNA remaining in the agarose plugs decreases exponentially for increasing radiation doses or particle fluences . From these curves cross sections for the induction of DSB after heavy ion irradiation have been determined . These results will be discussed in comparison to the results for cell survival.

Water Sci Technol, 1995, 31(5-6), 259 - 61
Comparative performance of Colisure (TM) and accepted methods in the detection of chlorine-injured total coliforms and E.coli; McFeters GA et al.; Studies were done to examine the comparability of Colisure (TM) and accepted reference methods to detect low numbers of total coliform bacteria and E.coli subjected to chlorine stress . Colisure (TM) is a medium designed to concurrently detect coliform bacteria and E.coli in drinking water by the specific action of beta-galactosidase (total coliforms) and beta-glucuronidase (E.coli) . The methods used to compare the performance of various media followed a protocol established by the USEPA . Samples (31) of sewage from six different regions of the US were treated with sufficient concentrations of chlorine (1.2-2.5mg/l) to reduce viability 1-3 logs (39% average injury) and diluted with drinking water to achieve ca . 3 viable coliforms/100ml . The mean log reductions in viable bacteria, determined with various media following disinfection of the 31 samples were: mEndo = 1.87 (TC), Colisure (TM) = 1.55 (TC), mTec = 3.63 (E.coli) and Colisure (TM) = 2.01 (E.coli) . When Colisure (TM) was compared with accepted methods to detect total coliforms in the dilute, disinfected samples, Colisure (TM) yielded results that were 1.6 times greater than LTB confirmed in BGLB at 28h . Colisure (TM) also detected 1.7 times greater levels of E.coli than LTB confirmed in EC/MUG at 28h . Sensitivity and specificity of Colisure (TM) were between 96 and 100% when positive and negative tests were verified . These findings indicate that Colisure (TM) is superior to certain accepted reference methods in the detection of chlorine-injured coliforms and E.coli under conditions that resemble contaminated drinking water.

Water Sci Technol, 1993, 27(3-4), 261 - 5
Chlorine injury and the comparative performance of Colisure (TM), ColiLert (TM) and ColiQuik (TM) for the enumeration of coliform bacteria and E.coli in drinking water; McFeters GA et al.; Several factors have stimulated interest in recently developed substrate specific media for the detection of coliform bacteria in water . This study compared the performance of Colisure (TM) (Millipore), ColiLert (TM) (Environetics) and ColiQuick (TM) (Hach) with accepted membrane filtration and MPN methodologies for the enumeration of total coliforms and E . coli in chlorinated water . The performance of all three media was compared, in MPN configuration, with LTB/MPN (confirmed) using a variety of drinking and source water samples, both with and without chlorination . The Cochran-Mantel-Haenszel test yielded statistical correlations between results obtained with each of the three new enzyme detection media and accepted reference methods for the detection of low numbers of total coliforms . Another series of tests compared the performance of Colisure with accepted methods (LTB/MPN confirmed with BGLB and EC-MUG) in the detection of total coliforms and E . coli in sewage-spiked samples simulating contaminated drinking water, using an USEPA/AWWA test protocol . The results demonstrated that Colisure detected these indicator bacteria with greater sensitivity than the accepted methods and that this difference increased between 24 and 28 hours of incubation . The results of this study collectively support the validity of the new enzyme detection method for the detection of low levels of coliform bacteria and E . coli in source water and contaminated drinking water.

Adv Space Res, 1992, 12(2-3), 65 - 8
Microdosimetric considerations of effects of heavy ions on E . coli K-12 mutants; Takahashi T et al.; The inactivation cross sections of E . coli K-12 recombination-deficient mutants, JC1553 (recA) and AB2470 (recB), for several MeV/u alpha-particles and N ions have been successfully analyzed by Katz's target theory in which radiosensitivity parameter E0 is assumed to be LET independent and equal to D37 for gamma-rays . For E . coli K-12 wild type, AB1157 (rec+, uvr+), however, it is impossible to interpret the inactivation cross section data by an LET-independent E0-value . In the latter case, as in the case of B . subtilis spore, it is necessary to assume that the radiosensitivity of the target for the core of a heavy ion is higher than that for delta-electrons . As well as Waligorski, Hamm and Katz's dose, the dose around the trajectory of an ion based on Tabata and Ito's energy deposition algorithm for electrons has been used in the course of analysis.

Mol Gen Mikrobiol Virusol, 2001, (3), 15 - 8
{Modulation of the cat gene expression in E . coli cells by simple (AC)20 repeats}; Pisarchik AV et al.; Probable relationship between modulation of gene expression by simple repeating sequences and competition capacity of the host was studied . (AC)20 repeat can both stimulate and inhibit the expression of chloramphenicol acetyl transferase gene expression in E . coli cells depending on this gene's position . Modulation of the gene expression by simple (AC)20 repeat and competitive capacity of bacteria containing this plasmid are closely related.

Nat Cell Biol, 2001 Sep, 3(9), 856 - 9
Enteropathogenic E . coli Tir binds Nck to initiate actin pedestal formation in host cells; Gruenheid S et al.; Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes infantile diarrhea worldwide . EPEC injects a bacterial protein, translocated intimin receptor (Tir), into the host-cell plasma membrane where it acts as a receptor for the bacterial outer membrane protein, intimin . The interaction of Tir and intimin triggers a marked rearrangement of the host actin cytoskeleton into pedestals beneath adherent bacteria . On delivery into host cells, EPEC Tir is phosphorylated on tyrosine 474 of the intracellular carboxy-terminal domain, an event that is required for pedestal formation . Despite its essential role, the function of Tir tyrosine phosphorylation has not yet been elucidated . Here we show that tyrosine 474 of Tir directly binds the host-cell adaptor protein Nck, and that Nck is required for the recruitment of both neural Wiskott-Aldrich-syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex to the EPEC pedestal, directly linking Tir to the cytoskeleton . Cells with null alleles of both mammalian Nck genes are resistant to the effects of EPEC on the actin cytoskeleton . These results implicate Nck adaptors as host-cell determinants of EPEC virulence.

Cell, 2001 Aug 24, 106(4), 429 - 41
Crystal structure of the processivity clamp loader gamma (gamma) complex of E . coli DNA polymerase III; Jeruzalmi D et al.; The gamma complex, an AAA+ ATPase, is the bacterial homolog of eukaryotic replication factor C (RFC) that loads the sliding clamp (beta, homologous to PCNA) onto DNA . The 2.7/3.0 A crystal structure of gamma complex reveals a pentameric arrangement of subunits, with stoichiometry delta':gamma(3):delta . The C-terminal domains of the subunits form a circular collar that supports an asymmetric arrangement of the N-terminal ATP binding domains of the gamma motor and the structurally related domains of the delta' stator and the delta wrench . The structure suggests a mechanism by which the gamma complex switches between a closed state, in which the beta-interacting element of delta is hidden by delta', and an open form similar to the crystal structure, in which delta is free to bind to beta.

Cell, 2001 Aug 24, 106(4), 417 - 28
Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E . coli DNA polymerase III; Jeruzalmi D et al.; The dimeric ring-shaped sliding clamp of E . coli DNA polymerase III (beta subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader gamma complex (homolog of eukaryotic Replication Factor C, RFC) . The delta subunit of the gamma complex binds to the beta ring and opens it . The crystal structure of a beta:delta complex shows that delta, which is structurally related to the delta' and gamma subunits of the gamma complex, is a molecular wrench that induces or traps a conformational change in beta such that one of its dimer interfaces is destabilized . Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the beta ring opens spontaneously once a dimer interface is perturbed by the delta wrench.

Cell Stress Chaperones, 2001 Jan, 6(1), 29 - 37
DnaK/DnaJ chaperone system reactivates endogenous E . coli thermostable FBP aldolase in vivo and in vitro; the effect is enhanced by GroE heat shock proteins; Kedzierska S et al.; Thermally aggregated, endogenous proteins in Escherichia coli cells form the S fraction, which is separable by sucrose density gradient centrifugation . To date, relatively little is known about the mechanisms of elimination of the heat-aggregated proteins from E . coli cells and the composition of the S fraction . We have identified several proteins of the S fraction using 2D-gel electrophoresis and microsequencing . A thermostable II class fructose-1,6-bisphosphate aldolase (Fda protein) appeared to be one of numerous proteins of the S fraction . Fda was purified from E . coli overproducer strain and used as a model substrate for investigation of the role of Hsps in prevention and repair of thermal denaturation of proteins both in vivo and in vitro . We found that the heat inactivation of Fda was reversible and that its reactivation in vivo and in vitro required mainly the assistance of the DnaK/DnaJ chaperone system . The dnaK756 and dnaJ259 mutations had a negative effect on the reactivation of thermally inactivated Fda . Moreover, we showed that the reactivation process in vitro was enhanced when GroEL/GroES were added together with DnaK/DnaJ . GroEL/GroES alone were inefficient in the resolubilization or reactivation of the heat-aggregated Fda . It is supposed that the denaturation of the thermostable Fda in vivo results rather from a temporary and transient deficit of Hsps than from the direct heat effect.

Biochemistry, 2001 Sep 4, 40(35), 10500 - 6
Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E . coli; Kol MA et al.; The mechanism by which phospholipids translocate (flop) across the E . coli inner membrane remains to be elucidated . We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane . As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E . coli phospholipids . Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane . Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C . The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation . In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop . For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG . In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE . A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.

Int J Biol Macromol, 2001 Aug 20, 29(2), 99 - 105
Unfolding and inactivation of monomeric superoxide dismutase from E . coli by SDS; Bozzi M et al.; The inactivation and the unfolding of the naturally monomeric Cu, Zn, superoxide dismutase from E . coli upon addition of sodium dodecylsulphate have been studied . In contrast to the bovine enzyme, CD, EPR, NMR spectroscopy and pulsed low resolution NMR measurements found an unfolding transition followed by inactivation of the enzyme . During this transition the active site becomes accessible to the bulk water . The unfolding is reversible and both, the tridimensional structure of the protein and the active site, can be restored upon dialysis . In addition, unfolding occurs without loss of metals in the solution.

Biotechniques, 2001 Aug, 31(2), 322 - 3, 326-8
Novel vectors for co-expression of two proteins in E . coli; Kholod N et al.; Two new vectors, pAC28 and pEGST, for the co-expression of recombinant genes in E . coli were developed . This two-plasmid system allows for an efficient expression and purification of large amounts of protein-protein complexes formed in bacterial cells . We have utilized this new system to express and isolate a stable complex of two human proteins, hematopoietic cell tyrosine phosphatase (HePTP) and mitogen-activated proteins kinase Erk2 . This approach is useful for biochemical and structural studies of protein-protein interactions.

Zhonghua Gan Zang Bing Za Zhi, 2001 Jul, 9 Suppl, 69 - 72
{The expression of HCV RNA polymerase in E.coli and the study of its solubility and antigenicity}; Hou L et al.; OBJECTIVE: To express HCV RNA polymerase and study its soluble condition and antigenicity . METHODS: We constructed expression vectors pQE-5B-Fl and pQE-5B-C21 and expressed them in E.coli (M15) . We analyzed their solubility in different conditions and purified soluble pQE-5B-C21 protein by Ni-NTA column, then detected its antigenicity by ELISA and western blot . RESULTS: We obtained the purified soluble pQE-5B-C21 protein in the induction conditions of 18 degrees C and the protein was proved to be of good antigenicity by ELISA and western blot . CONCLUSIONS: The RNA polymerase of HCV expressed in E.coli has good solubility and antigenicity.

Mutat Res, 2001 Sep 1, 480-481, 77 - 84
Contribution of E . coli AlkA, TagA glycosylases and UvrABC-excinuclease in MMS mutagenesis; Grzesiuk E et al.; MMS, an S(N)2 alkylating agent, is a moderate inducer of SOS mutagenesis and adaptive response . Our previous studies have shown that transient starvation of Escherichia coli AB1157argE3 strain causes a decrease of MMS-induced argE3-->Arg(+) reversions and this decrease is accompanied by the disappearance of the Fpg protein sensitive sites on plasmids isolated from MMS-treated and subsequently starved bacteria . This suggests that in such cells the mutation frequency decline (MFD) repair takes place . Here, we study the relation between MMS-induced mutagenesis as well as mutation frequency decline during starvation, and the repair of alkylated bases and AP-sites by base and nucleotide excision repair systems . In the AB1157alkA(-) strain, MMS-induced mutagenesis was over five-fold higher than in the wild type strain and no MFD repair occurred during starvation . Surprisingly, the lack of TagA glycosylase diminished MMS mutagenesis and accelerated the MFD effect . However, in double tagA(-)alkA(-) mutant, the frequency of Arg(+) reversions increased over 10-fold during 60 min of aminoacid starvation after MMS-treatment . Lack of the uvrA gene function did not affect the MMS-induced mutation rate and MFD in AB1157alkA(+)tagA(+) . Starvation of MMS treated AB1157tagAalkAuvrA triple mutant caused a decrease of mutation frequency almost to the level of spontaneous mutation rate . Examination of the repair of 3-MeAde, 7-MeGua and AP sites during starvation using repair glycosylases and plasmids isolated from MMS-treated and starved bacteria revealed that in E . coli uvr(+) but tagAalkA strain, neither 3-MeAde nor 7-MeGua were repaired during 60 min starvation and these persistent lesions could be responsible for the induction of the SOS system and an increase in mutation rate during starvation . In the triple tagAalkAuvrA mutant the repair of 3-MeAde, 7-MeGua and AP sites was carried out effectively and this could explain the observed decrease in the mutation rate during starvation . These results suggest that only in the absence of the "first choice" repair enzymes TagA, AlkA glycosylases and UvrABC excinuclease, a third error-free repair system of alkylated bases is activated . In the absence of only TagA and AlkA glycosylases, UvrABC excinuclease mediates activation of the SOS response, and this results in an increase of mutagenesis induced by the presence of alkylated bases in DNA.

Mutat Res, 2001 Sep 1, 480-481, 71 - 5
The antimutator phenotype of E . coli mud is only apparent and results from delayed appearance of mutants; Schaaper RM et al.; Antimutator strains are strains that have a lower mutation rate than the wild-type strain . We have reexamined the properties of one reported antimutator strain of Escherichia coli, termed mud {Mol . Gen . Genet . 153 (1977) 87} . This strain contains a temperature-sensitive mutation in the purB gene, leading to adenine-dependent growth at higher temperature . When grown at permissive or semi-permissive temperature in the absence of adenine it displays large reductions in the number of both spontaneous and mutagen-induced mutants (e.g . several hundred-fold for valine-resistant mutants) . However, our studies show that strains containing the purB allele generate mutations at the same level as the wild-type strain, and that the apparent antimutator effect is the consequence of the delayed appearance of mutants on the selective plates . This delay likely results from the combined stress exerted by the adenine deficiency and the presence of the selective agent (i.e . valine).

Biochemistry, 2001 Aug 21, 40(33), 9968 - 76
Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E . coli transhydrogenase; Althage M et al.; The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport . In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction . Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed . Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266 . A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge . The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions . Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values . It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.

J Mol Biol, 2001 May 25, 309(1), 255 - 66
E . coli 5'-nucleotidase undergoes a hinge-bending domain rotation resembling a ball-and-socket motion; Knofel T et al.; Structures of nine independent conformers of E . coli 5'-nucleotidase (5'-NT) have been analyzed using four different crystal forms . These data show that the two-domain protein undergoes an unusual 96 degrees hinge-bending domain rotation . Structures of the open and closed forms with substrates and inhibitors reveal that the substrate moves by approximately 25 A with the large domain rotation into the catalytic site . The domain motions derived from a comparison of the nine conformations agree well with motions obtained from a normal mode analysis in that all independent domain rotations are around axes that are roughly located in the plane which includes the domain centers and the hinge . Two residues, Lys355 and Gly356, form the core of the hinge region and undergo a large change of the main-chain torsion angles . The hinge-bending movement observed for 5'-nucleotidase differs markedly from a classical hinge-bending closure motion which involves an opening of the substrate or ligand-binding cleft between two domains . In contrast, the movement observed in 5'-nucleotidase resembles that of a ball-and-socket joint . The smaller C-terminal domain rotates approximately around its center such that the residues at the domain interface move in a sliding motion along the interface . Few direct interdomain contacts and a layer of water molecules between the two domains facilitate the sliding motion.

Cell Microbiol, 2001 Aug, 3(8), 499 - 510
The enterohaemorrhagic Escherichia coli (serotype O157:H7) Tir molecule is not functionally interchangeable for its enteropathogenic E . coli (serotype O127:H6) homologue; Kenny B; A major virulence determinant of enteropathogenic Escherichia coli (EPEC) is the Tir molecule that is translocated into the plasma membrane where it orchestrates cytoskeletal rearrangements . Tir undergoes several phosphorylation events within host cells, with modification on a tyrosine essential for its actin-nucleating function . The EHEC (serotype O157:H7) Tir homologue is not tyrosine phosphorylated implying that it uses an alternative mechanism to nucleate actin . This is supported in this study by the demonstration that EHEC Tir is unable to functionally substitute for its EPEC homologue . Like EPEC, the EHEC Tir molecule is phosphorylated within host cells, with the actin-nucleating dysfunction correlated to an altered modification profile . In contrast to EHEC Tir, the EPEC Tir molecule mediated actin nucleation whether delivered into host cells by either strain . Thus, it would appear that EHEC encodes specific factor(s) that facilitate the correct modification of its Tir molecule within host cells . Domain-swapping experiments revealed that the N-terminal, alpha-actinin binding, Tir domains were functionally interchangeable, with both the actin-nucleating dysfunction and altered modification profiles linked to the EHEC C-terminal Tir domain . This tyrosine-independent modification process presumably confers an advantage to EHEC O157:H7 and may contribute to the prevalence of this strain in EHEC disease . The presented data are also consistent with EPEC and EHEC sharing non-phosphotyrosine phosphorylation event(s), with an important role for such modifications in Tir function . An EHEC-induced phosphotyrosine dephosphorylation activity is also identified.

Water Res, 2001 Sep, 35(13), 3085 - 8
The occurrence of E . coli O157:H7 in South African water sources intended for direct and indirect human consumption; Muller EE et al.; The occurrence of Escherichia coli O157:H7 in selected water samples in South Africa was investigated . The chromogenic Rainbow agar O157 medium designed for the rapid identification of E . coli O157:H7 was used for the detection of these organisms in various river-water samples in the Vaal Barrage Reservoir drainage basin of South Africa . A total of 204 samples were obtained from 15 sites where water was used for direct and indirect human consumption . Samples were filtered through Gelman filter-units and incubated on Rainbow agar O157 which produced different colours according to the bacterial chromogenic properties . Six hundred and sixty-three suspected E . coli O157:H7 colonies, with colours ranging between dark blue, grey and black, were subcultured onto sorbitol-MacConkey agar and screened for different virulence factors specific for E . coli O157:H7 and agglutination with anti-E . coli O157 antiserum . The results indicated that none of the suspected colonies contained all of the virulence factors necessary to classify them as E . coli O157:H7 . None of these organisms agglutinated with antisera against E . coli O157 . The probability of being infected with E . coli O157:H7 from direct or indirect consumption of these river water sources is therefore low . Some samples did, however, contain enterohaemorrhagic E . coli virulence properties, such as Stx1, Stx2 and enterohaemolysin, which might impose a health risk if ingested.

Biotechnol Prog, 2001 Jul-Aug, 17(4), 624 - 8
Cell growth and by-product formation in a pyruvate kinase mutant of E . coli; Zhu T et al.; In this paper, we report on the analysis of acid formation in an E . coli pyk mutant . The results demonstrate that acid formation is insignificant for both the wild-type and the mutant at low glucose concentrations . However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild-type . This substantial reduction in acids is accompanied by an increase in CO(2) production . Moreover, unlike the B . subtilis pyk mutant, the E . coli pyk mutant did not show a substantial increase in the PEP pool.

Neurosci Lett, 2001 Aug 10, 308(3), 149 - 52
Impaired retrograde axonal transport of adenovirus-mediated E . coli LacZ gene in the mice carrying mutant SOD1 gene; Murakami T et al.; A replication-defective recombinant adenoviral vector containing E . coli lacZ gene was injected into the gastrocnemius muscles of transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) gene and non-transgenic wild-type mice at 40 weeks of age . After 60 and 90 h of the injection, lacZ staining was observed at the distal ends of the sciatic nerves in both mice groups, with the number and the distances greatly reduced in the transgenic mice . Mean velocities of retrograde transport for lacZ was estimated to be 2.1 and 0.05 mm/24 h in non-transgenic and transgenic mice, respectively . These results indicate that the retrograde axonal transport of foreign gene product is impaired in the mice model for familial amyotrophic lateral sclerosis.

Science, 2001 Jul 27, 293(5530), 705 - 8
Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E . coli; Kuroda A et al.; Inorganic polyphosphate (polyP), a polymer of hundreds of phosphate (Pi) residues, accumulates in Escherichia coli in response to stresses, including amino acid starvation . Here we show that the adenosine 5'-triphosphate-dependent protease Lon formed a complex with polyP and degraded most of the ribosomal proteins, including S2, L9, and L13 . Purified S2 also bound to polyP and formed a complex with Lon in the presence of polyP . Thus, polyP may promote ribosomal protein degradation by the Lon protease, thereby supplying the amino acids needed to respond to starvation.

J Photochem Photobiol B, 2001 Jul, 60(2-3), 136 - 42
Induction of phr gene expression in E . coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation; Kohli R et al.; We have observed that He-Ne laser irradiation of E . coli strain KY706/pPL-1 leads to induction of photolyase gene, phr . The magnitude of induction was found to depend on the He-Ne laser fluence, fluence rate and post-irradiation incubation period in the nutrient medium . The optimum values for fluence and fluence rate were 7x10(3) J/m(2) and 100 W/m(2), respectively, and the induction of phr gene was observed to saturate beyond an incubation period of approximately 2 h . Experiments carried out with singlet oxygen quenchers and with D(2)O suggest that the effect is mediated via singlet oxygen . Photoreactivation studies carried out after UVC exposure of both the He-Ne laser-exposed as well as unexposed cells showed a larger surviving fraction in the He-Ne laser pre-irradiated cells . This can be attributed to He-Ne laser irradiation-induced induction of phr expression . However, since even without photoreactivating light He-Ne laser pre-irradiated cells show higher survival against UVC radiation it appears that He-Ne laser irradiation induces both light-dependent as well as dark DNA repair processes.

Mol Genet Genomics, 2001 Jun, 265(4), 683 - 6
The RadB protein from Pyrococcus does not complement E . coli recA mutations in vivo; Inwood W et al.; A previous publication claimed that the radB gene called Pk-REC from Pyrococcus furiosus complemented an E . coli recA mutation . We found that a sequencing error had led to the test of a mutant form of Pk-REC . The wild-type radB gene from P . furiosus cloned in a similar expression vector to the mutant Pk-REC also appeared to complement an E . coli recA mutation . However, the cloned P . furiosus gdh (glutamate dehydrogenase) gene showed the same activity . We therefore concluded that overexpression of any protein can produce an artificial growth inhibition or stationary phase in recA mutant cells, which allows cells to recover from UV damage due to the action of repair systems that do not require RecA-like activity.

Bioinformatics, 2001 Jul, 17(7), 622 - 33
Conformational model for binding site recognition by the E.coli MetJ transcription factor; Liu R et al.; MOTIVATION: Current methods for identifying sequence specific binding sites in DNA sequence using position specific weight matrices are limited in both sensitivity and specificity . Double strand DNA helix exhibits sequence dependent variations in conformation . Interactions between macromolecules result from complementarity of the two tertiary structures . We hypothesize that this conformational variation plays a role in transcription factor binding site recognition, and that the use of this structure information will improve the predictive power of transcription factor binding site models . RESULTS: Conformation models for the sequence dependence of DNA helix distortion have been developed . Using our conformational models, we defined a tertiary structure template for the met operon repressor MetJ binding site . Both naturally occurring sites and precursor binding sites identified through in vitro selection were used as the basis for template definition . The conformational model appears to recognize features of protein binding sites that are distinct from the features recognized by primary sequence based profiles . Combining the conformational model and primary sequence profile yields a hybrid model with improved discriminatory power compared with either the conformational model or sequence profile alone . Using our hybrid model, we searched the E.coli genome . We are able to identify the documented MetJ sites in the promoter regions of metA, metB, metC, metR and metF . In addition, we find several novel loci with characteristics suggesting that they are functional MetJ repressor binding sites . Novel MetJ binding sites are found upstream of the metK gene, as well as upstream of a gene, abc, a gene that encodes for a component of a multifunction transporter which may transport amino acids across the membrane . The false positive rate is significantly lower than the sequence profile method . AVAILABILITY: The programs of implementation of this algorithm are available upon request . The list of crystal structures used for compiling the mean base step parameters of DNA is available by anonymous ftp at http://stateslab.wustl.edu/pub/helix/StructureList.

Cell, 2001 Jun 15, 105(6), 733 - 43
Structural basis of the interaction of the pyelonephritic E . coli adhesin to its human kidney receptor; Dodson KW et al.; PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney . The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide . Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin . The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis . These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E . coli for the human kidney and is critical to the pathogenesis of pyelonephritis.

Mol Cell, 2001 Jun, 7(6), 1337 - 43
Topological regulation of cell division in E . coli . spatiotemporal oscillation of MinD requires stimulation of its ATPase by MinE and phospholipid; Hu Z et al.; Topological regulation of cell division in E . coli requires positioning a cell division inhibitor, MinC, at the poles of the cell, thus restricting the potential for division to midcell . This positioning is achieved through a rapid oscillation of MinC from pole to pole, a process requiring MinD and MinE . However, the mechanistic basis for this oscillation is not known . Here we report that MinE stimulates MinD ATPase activity, but only in the presence of phospholipid vesicles . Analysis of MinE mutants demonstrates that this stimulation is required for MinD oscillation and suggests that the level of stimulation determines the period of the oscillation . A model is presented in which the requirements for the MinD ATPase contribute spatial and temporal inputs that provide the mechanistic basis for the oscillation.

J Mol Biol, 2001 Jul 6, 310(2), 485 - 98
High enzymatic activity and chaperone function are mechanistically related features of the dimeric E . coli peptidyl-prolyl-isomerase FkpA; Ramm K et al.; We have recently described the existence of a chaperone activity for the dimeric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escherichia coli that is independent of its isomerase activity . We have now investigated the molecular mechanism of these two activities in vitro in greater detail . The isomerase activity with a protein substrate (RNaseT1) is characterized by a 100-fold higher k(cat)/K(M) value than with a short tetrapeptide substrate . This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding site that is distinct from the active site . The chaperone activity is also mediated by interaction of folding and unfolding intermediates with a binding site that is most likely identical to the polypeptide-binding site which enhances catalysis . Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-binding site . Only the isomerase activity, but not the chaperone activity, with the substrate citrate synthase can be inhibited by FK520 . Experiments with the isolated domains of FkpA imply that both the isomerase and the chaperone site are located on the highly conserved FKBP domain . The additional amino-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneously interact with a protein, and this is required for full catalytic activity .

Mutat Res, 2001 Jul 1, 478(1-2), 191 - 7
Bisulfite-induced cytosine deamination rates in E . coli SSB:DNA complexes; Lough J et al.; E . coli single-stranded binding protein (SSB) has been examined for its ability to modulate bisulfite-induced cytosine deamination rates in single-stranded DNA (ssDNA) . We used a lacZ alpha-complementation reversion assay to detect C-->U rates at a single codon in M13mp2 DNA, whether in free ssDNA or in an SSB:ssDNA complex . When incubated at 37 degrees C, the average bisulfite-induced reversion rate constant was four-fold less in SSB:ssDNA complexes than in ssDNA, at a single codon . Across a 250 base pair target and over 23 scorable C-->U sites, the forward rate constant was 4.9-fold less in SSB:ssDNA complexes than in ssDNA alone . After treatment with N-uracil glycosylase, ssDNA incubated with bisulfite had reversion frequencies at the background rate of ssDNA incubated without bisulfite, indicating that virtually all mutations scored were due to C-->U events . The decrease in cytosine deamination rates occurred both in a single codon and over a 250 bp target, indicating that interactions between SSB and ssDNA reduce bisulfite-catalyzed mutations . The structural role of SSB is well recognized in multiple cellular processes; SSB can also function to minimize bisulfite-induced ssDNA mutations.

Curr Microbiol, 2001 Aug, 43(2), 89 - 92
An E . coli 5S rRNA deletion mutant useful for the study of 5S rRNA structure/function relationships; Ammons D et al.; We report on the construction of a novel strain of E . coli that can be useful for studies on the structure/function relationship of 5S rRNAs . The bacterial strain is deficient in six of the eight naturally occurring 5S rRNA genes (operons B, D, H, G, E) and demonstrates a greatly reduced growth rate that can be compensated by the plasmid-encoded expression of 5S rRNA . The relatively large difference in growth rate between compensated and non-compensated mutants provides the basis for a quick and simple assaying system for both the evaluation and mass screening of divergent 5S rRNA sequences for function . We describe the construction of the 5S rRNA deletion mutant BDHGE and characterize the usefulness and limitations of the system for evaluating structure/function relationships of 5S rRNA sequence.

Biochemistry, 2001 Jun 12, 40(23), 6713 - 9
Activation of class III ribonucleotide reductase from E . coli . The electron transfer from the iron-sulfur center to S-adenosylmethionine; Padovani D et al.; The anaerobic ribonucleotide reductase (ARR) from E . coli is the prototype for enzymes that use the combination of S-adenosylmethionine (AdoMet) and an iron-sulfur center for generating catalytically essential free radicals . ARR is a homodimeric alpha2 protein which acquires a glycyl radical during anaerobic incubation with a {4Fe-4S}-containing activating enzyme (beta) and AdoMet under reducing conditions . Here we show that the EPR-active S = 1/2 reduced {4Fe-4S}+ cluster is competent for AdoMet reductive cleavage, yielding 1 equiv of methionine and almost 1 equiv of glycyl radical . These data support the proposal that the glycyl radical results from a one-electron oxidation of the reduced cluster by AdoMet . Reduced protein beta alone is also able to reduce AdoMet but only in the presence of DTT . However, in that case, 2 equiv of methionine per reduced cluster was formed . This unusual stoichiometry and combined EPR and Mossbauer spectroscopic analysis are used to tentatively propose that AdoMet reductive cleavage proceeds by an alternative mechanism involving catalytically active {3Fe-4S} intermediate clusters.

Neurol Sci, 2000, 21(5 Suppl), S983 - 4
A novel mtDNA mutation in the ATPase6 gene studied by E . coli modeling; Carrozzo R et al.; This study aimed to understand the pathogenesis of a new mtDNA-related etiology of Leigh syndrome . We identified the T9176G mutation as the molecular basis of Leigh syndrome in a child and looked for alterations in cellular ATP production . We then modeled the new mtDNA mutation in E . coli and analyzed ATP synthesis, hydrolysis, and the ability of the mutated enzyme to pump protons . Our results suggest that the T9176G change results in a novel, fully assembled enzyme which inhibits the holoenzyme probably by blocking the proton pathway.

Mutat Res, 2001 May 31, 492(1-2), 91 - 7
Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E . coli; Ohta T et al.; Ethidium bromide (EtBr) and SYBR Green I are nucleic acid gel stains and used commonly in combination with UV-illumination . EtBr preferentially induces frameshift mutations but only in the presence of an exogenous metabolic activation system, while SYBR Green I is a very weak mutagen that induces frameshift mutations . We found that EtBr and SYBR Green I, without an added metabolic activation system, strongly potentiated the base-substitution mutations induced by UV-irradiation in E . coli B/r WP2 cells . Each DNA stain alone showed no mutagenicity to the strain . Base-substitutions induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and 4-nitroquinoline-1-oxide were similarly potentiated by EtBr and SYBR Green I . SYBR Green I had a much greater effect . No enhancing effects were observed on mutations induced by mitomycin C, cisplatin, transplatin, cumene hydroperoxide, base analogs, and alkylating agents . Another DNA stain, acridine orange, showed similar enhancing effects on UV- and MX-mutagenicity, but no effect was observed for 4',6-diamidino-2-phenylindole (DAPI) . UV- and MX-induced mutations in E . coli WP2s (uvrA), which is defective in nucleotide excision repair activity, were not potentiated by the addition of EtBr, SYBR Green I, or acridine orange . Those nucleic acid stains might inhibit the nucleotide excision repair of DNA damaged by UV and MX treatment.

Structure (Camb), 2001 May 9, 9(5), 439 - 50
The crystal structure of E . coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily; von Delft F et al.; BACKGROUND: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis . It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction . RESULTS: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E . coli . The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution . The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid . CONCLUSIONS: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily . This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex.

Arch Biochem Biophys, 2001 Jan 15, 385(2), 311 - 21
The kinetic and spectral characterization of the E . coli-expressed mammalian CYP4A7: cytochrome b5 effects vary with substrate; Loughran PA et al.; The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins . CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E . coli by adding the first 10 codons of CYP17alpha producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies . CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min(-1) and corresponding Km values of 74.5, 27, and 16.7 microM, respectively, in the presence of cytochrome b5 . In the absence of cytochrome b5, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min(-1) and corresponding Km values of 3.9 and 33 microM, respectively . Arachidonate was not metabolized in the absence of cytochrome b5 . Saturation kinetics studies performed with heme-depleted cytochrome b5 (apo cytochrome b5) yielded turnover numbers of 118 and 74 min(-1) and Km values of 74 and 25 microM with laurate and myristate, respectively, indicating that cytochrome b5 is not involved in electron transfer but rather plays a conformational role . Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (KS) in the absence and presence of cytochrome b5 (13 and 43 microM, respectively) . In stopped-flow kinetics experiments, the flavin reduction (approximately 90 s(-1)) and heme reduction (approximately 9 s(-1)) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b5 . Estimations of the rate of CPR (0.3 s(-1)) or cytochrome b5 (9.1 s(-1)) binding with CYP4A7 were also determined.

Arzneimittelforschung, 2001, 51(4), 332 - 8
Double-blind randomised placebo-controlled phase III study of an E . coli extract plus 5-fluorouracil versus 5-fluorouracil in patients with advanced colorectal cancer; Unger C et al.; The primary aim of this study was to evaluate the toxicity (mucositis, diarrhea and leucopenia) of a therapy with 5-fluorouracil (CAS 51-21-8; 5-FU) plus an E . coli extract (LC-Extract, Laves coli extract, Colibiogen inject, cell-free soluble fraction from lysed E . coli, Laves strain) in comparison with 5-FU plus placebo . Secondary endpoints included general toxicity, response rate according to WHO, survival time and quality of life . 164 patients with advanced colorectal cancer were enrolled in this randomised, placebo-controlled, double-blind, multicenter phase III study . The treatment consisted of 0.167 ml/kg/d LC-Extract or placebo followed by 500-750 mg/m2/d 5-FU on five consecutive days, repeated every three weeks for up to six treatment cycles . 158 (77 verum, 81 placebo) patients were evaluable for toxicity, 144 (72 verum, 72 placebo) evaluable for response . The therapy with LC-Extract was well tolerated . Adverse events that occurred during the study were mainly judged as 5-FU- or tumor-related . Toxicity from treatment with 600 mg/m2/d 5-FU in both treatment groups was very low . After treatment with 750 mg/m2/d 5-FU patients in the placebo-group experienced a higher CTC toxicity than in the LC-Extract groups . Remission rate and survival time showed a slight trend in favour of LC-Extract . These results suggest a positive benefit-risk ratio of the additional application of LC-Extract to 5-FU in the treatment of advanced colorectal cancer especially for administration of high doses of 5-FU.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1998 Aug, 20(4), 279 - 84
{The expression study of human stem cell factor (hSCF) in E . coli}; Mao H et al.; OBJECTIVE: To improve the expression level of recombinant human stem cell factor (rhSCF) . METHODS: With PCR techniques and synthesized oligonucleotide as primers, the number of nucleotides between the SD sequence and the starting codon ATG of human stem cell factor cDNA was altered, and the preferable codons of E . coli in the N-terminal amino acid sequence were selected . RESULTS: SDS-PAGE electrophoresis indicated that the ratio of expressed rhSCF to total E . coli proteins increased from 12% to 40% by thermal inducing . With DNA sequencing, the reading frame of the gene was proved to be correct . The sequence of 17 N-terminal amino acids of purified recombinant hSCF has been proved to be identical to the natural hSCF, except the first amino acid-Met . CONCLUSIONS: These studies suggest that the 5'-flanking region of hSCF cDNA plays an important role in its gene expression.

J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 385 - 93
Regulation of E . coli glycogen phosphorylase activity by HPr; Seok YJ et al.; Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients . One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS) . Many proteins interact with and are regulated by PTS components in bacteria . Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS . HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown . The evolution of the allosteric regulation of E . coli GP by HPr is compared to that of other phosphorylases.

J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 347 - 54
Three-dimensional structures of protein-protein complexes in the E . coli PTS; Peterkofsky A et al.; The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport . The soluble proteins of the glucose transport pathway also function as regulators of diverse systems . The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy . The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIA(Glc) have been elucidated . An analysis of the binding interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions . Similarly, a common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase . Thus, there is a common motif for the protein-protein interactions characteristic of the PTS.

Lung, 2000 Nov-Dec, 178(6), 331 - 40
Attenuation of live E . coli-induced acute lung injury by X-ray irradiation in guinea pigs; Ishizaka A et al.; Irradiation is suspected to injure inflammatory cells, such as neutrophils and mononuclear phagocytes, cells known to contribute to the development of acute lung injury (ALI) . This study examined whether preexposure to x-ray irradiation modifies ALI induced by E . coli injected intravenously in guinea pig . Thirty animals were divided into two control and two irradiated subgroups: the first control group received saline only (n = 8), and the second control group received E . coli, 2 x 10(9)/kg body weight, suspended in saline (n = 6), IV . The first irradiated group received a single 12-Gy dose + saline (n = 6), and the second irradiated group received a single 12-Gy dose + E . coli (n = 10) . The lung wet-to-dry-weight ratio (W/D) and 125I-albumin lung tissue/plasma ratio (T/P) were measured as markers of lung injury . W/D was significantly higher in the control E . coli group than in the other groups . T/P in the control E . coli group was also increased compared with T/P measured in the other groups . In the control E . coli group, a marked increase in bronchoalveolar lavage (BAL) neutrophils was observed compared with the control saline group . However, no significant difference in BAL neutrophil counts was observed between the control and irradiated E . coli groups . In contrast, BAL macrophages were significantly reduced in the irradiated E . coli groups compared with the control E . coli group . These findings suggest that x-ray irradiation attenuates E . coli-induced ALI in guinea pigs, an effect explained, at least in part, by a reduction in the number of alveolar macrophages.

Chin Med Sci J, 1997 Dec, 12(4), 229 - 31
The development of monoclonal antibody against rhTNF and its curative effects on E . coli infected mice; Guo X et al.; Fifteen hybridoma cell lines producing monoclonal antibodies (McAb) against recombinant human tumor necrosis factor alpha (rhTNFa) have been established by fusing SP 2/0 cells with spleen cells from a BALB/c mouse immunized with rhTNFa . Following J . M . Davis's Works, semi-solid medium was used for initial cloning . Five of them were studied further . Their main chromosome numbers range were 96 to 105, all of them were IgG1 subclass . The affinities of these McAbs were estimated to be 1.25 x 10(8) mol/L, 1.12 x 10(8) mol/L, 2.34 x 10(8) mol/L, 8.55 x 10(7) mol/L, 1.04 x 10(8) mol/L, respectively . Two groups of mice challenging with E . Coli (10(7) organisms), one group treated with 2 mg/kg anti-TNF monoclonal antibody, the other did not . There was a higher survival rate in treated group, the serum TNF level was significantly lower too, and the untreated mice had severe pathologic changes in viscera.

FEMS Microbiol Lett, 2001 May 15, 199(1), 61 - 6
A subtractive hybridisation analysis of genomic differences between the uropathogenic E . coli strain 536 and the E . coli K-12 strain MG1655; Janke B et al.; Suppression subtractive hybridisation (SSH) was performed to identify genomic differences between the uropathogenic Escherichia coli strain 536 and the non-pathogenic E . coli K-12 strain MG1655 . In total, 22 DNA fragments were isolated which were specific for strain 536 . Five of these fragments showed homology to known virulence determinants and four fragments matched genes for lipopolysaccharide (LPS) or capsule biosynthesis and a siderophore receptor . Seven fragments did not show any homology to known genes . These fragments may represent parts of putative pathogenicity islands (PAIs) . Whereas two fragments were highly specific for uropathogenic E . coli (UPEC), the other fragments could also be detected among the other tested wild-type strains.

Mutat Res, 2001 Jun 5, 486(1), 11 - 9
Expression of E . coli RecA targeted to mitochondria of human cells; Paul R et al.; Mitochondrial DNA integrity is ensured by several nuclear-encoded proteins in vertebrates, and a number of mtDNA alterations in human diseases, including deletions and duplications, have been suspected to result from errors in the mitochondrial recombination pathway . However, the presence of the latter system is still a matter of controversy as RecA proteins display various functions in vitro . In Escherichia coli, RecA plays a central role in homologous recombination by pairing and transferring a single strand to a homologous duplex DNA . To address indirectly the issue of a mitochondrial recombination pathway in vivo, we have constructed a chimeric gene containing an N terminal mitochondrial targeting sequence and the E . coli RecA gene . Cells were transfected by the recombinant plasmid, then tested for their mtDNA repair upon bleomycin treatment . We found an increased repair rate of the mitochondrial DNA in cells expressing RecA as compared to control cells . These results indicate that the transfected cells display an improved mtDNA repair replication pathway due to the exogenous RecA, likely in synergy with an endogenous rate-limiting mitochondrial recombination pathway.

Vet J, 2001 May, 161(3), 269 - 79
E . coli nitroreductase/CB1954: in vitro studies into a potential system for feline cancer gene therapy; Blackwood L et al.; Investigations were carried out to identify a suitable prodrug activating system for feline gene therapy with the eventual aim of treating feline thyroid disease and feline neoplasia . The E . coli nitroreductase (NTR)/CB1954 prodrug activating system was evaluated in vitro in feline cells by transient transfection with a nitroreductase expressing construct and subsequent treatment with the prodrug CB1954 . The feline cells successfully expressed E . coli nitroreductase, which was able to activate the prodrug CB1954 resulting in cytotoxicity to both transformed and adjacent cells (a bystander effect) in vitro . In the absence of nitroreductase, CB1954 was non-toxic to feline cells . In addition, the nitroreductase gene was expressed in rat thyroid cells under the control of the cell type specific feline thyroglobulin promoter . This paper demonstrates that the E . coli nitroreductase/CB1954 system may be suitable for in vivo feline gene therapy, and further investigations are warranted.

Biochemistry, 2001 Feb 20, 40(7), 2075 - 9
pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E . coli in planar lipid bilayers; Das S et al.; Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH . This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations . Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity . PHB/polyP complexes, isolated from E . coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH . When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5 . However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure . Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH . Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH . Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH . The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel.

Mol Cell Endocrinol, 2001 Apr 25, 175(1-2), 41 - 56
Ovine caveolin-1: cDNA cloning, E . coli expression, and association with endothelial nitric oxide synthase; Chen D et al.; Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obligatory role in regulating the activity of endothelial nitric oxide (NO) synthase (eNOS) . We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy . To test this hypothesis in the sheep model, we isolated the full-length cDNA of ovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental artery endothelial cells . Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced . Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from approximately 2.1 to 2.7 kb . Northern analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of approximately 2.1 and 2.7 kb, respectively . Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC . Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a approximately 1.6-2.1 kb 3'-untranslated region . Database searches with oCav-1 cDNA revealed that the coding region of mammalian Cav-1 genes is highly conserved . We prepared a recombinant full-length oCav-1 protein in which six consecutive histidine residues were tagged at the end of its COOH-terminus and developed a {His}6-tagged oCav-1 'pull-down assay' for studying the association of eNOS with Cav-1 . Incubation of exogenous {His}6-tagged oCav-1 with resting UAEC extracts led to the formation of a {His}6-tagged oCav-1-eNOS complex . In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) peptide, but not a mutated CSD peptide, {His}6-tagged oCav-1 associated eNOS was dose (0-10 microM)-dependently inhibited . eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co-existed with Cav-1 during the isolation of caveolae membranes . Because dissociation of eNOS from Cav-1 is required for the activation of eNOS, eNOS association with Cav-1 in UAEC suggests an important role of Cav-1 in regulating UAEC production of NO and possibly NO-mediated uterine vasodilatation.

J Ocul Pharmacol Ther, 2001 Feb, 17(1), 93 - 9
Bovine eye 1-Cys peroxiredoxin: expression in E . coli and antioxidant properties; Peshenko IV et al.; Peroxiredoxins constitute a molecular family of novel antioxidant proteins and are distributed broadly in non-mammalian and mammalian tissues, including the eye . In this study, a recombinant bovine eye 1-Cys peroxiredoxin (BRPrx) was expressed in Escherichia coli (E . coli) . The recombinant protein protected glutamine synthetase from oxidative damage caused by a metal ion-catalyzed oxidation system (ascorbate/Fe3+/O2) in the presence of dithiothreitol as an electron donor . The protector activity of BRPrx is attributed to its peroxidase activity exhibited in the presence of dithiothreitol . Both hydrogen peroxide and short chain hydroperoxides are substrates for the protein . Glutathione could not support antioxidant properties of the recombinant protein . The antioxidant activity of BRPrx in the glutamine synthetase protection assay was as high as the activity of catalase and about one order of magnitude lower than that of selenium glutathione peroxidase . These results support the premise that Prx is an important component of the antioxidant defense system in eye tissues.

Biotechnol Bioeng, 2001 Jun 5, 73(5), 406 - 11
Integrated flow-injection processing for on-line quantification of plasmid DNA during cultivation of E . coli; Nandakumar MP et al.; An integrated flow-injection processing (FIP) system for the quantification of plasmids during cultivation is described . The system performs on-line sampling, cell lysis, and quantification of plasmids in an integrated manner during cultivation of E . coli . The system was operated by using a miniaturized expanded-bed column which can be used for handling samples containing cells and cell debris without interfering with the binding analysis . Two types of detectors (one measuring UV absorbance at 254 nm and a fluorometer) are used for on-line plasmid detection . The system was developed using standard solutions and it was successfully applied in monitoring plasmid contents during a cultivation of E . coli .

Genes Cells, 2001 Apr, 6(4), 279 - 90
Induction of CspA, an E . coli major cold-shock protein, upon nutritional upshift at 37 degrees C; Yamanaka K et al.; BACKGROUND: The synthesis of CspA, the major cold-shock protein of Escherichia coli, is dramatically induced upon cold shock . It was recently reported that there is massive presence of CspA under nonstress conditions, and it is thus claimed that CspA as the cold-shock protein is a misnomer . RESULTS: Here, we re-examined and confirmed that CspA is induced upon culture dilution at 37 degrees C . However, its induction level is one-sixth of the cold-shock-induced level, clearly indicating that the major stress that induces CspA is cold shock . It was further found that CspA induction can be achieved not only by culture dilution but also by the simple addition of nutrients, and that it was almost completely abolished in the presence of rifampicin or nalidixic acid . Nutritional upshift causes the induction of only CspA but not other cold-shock-inducible CspA homologues . The amount of cspA mRNA rapidly and transiently increased by culture dilution, but its stability was not significantly changed . CONCLUSIONS: These results suggest that CspA is a nutritional-upshift stress protein as well as a cold-shock stress protein, and that CspA induction following nutritional upshift may be due to transcriptional activation.

J Protein Chem, 2000 Nov, 19(8), 685 - 92
The proteolytic activity of the recombinant cryptic human fibronectin type IV collagenase from E . coli expression; Schnepel J et al.; Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain . The enzyme could be generated and activated in the presence of Ca2+ from the purified 70-kDa pFN fragment produced by cathepsin D digestion . In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E . coli . The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, alpha- and beta-casein, insulin b-chain, and a synthetic Mca-peptide . In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates . Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2.

Clin Nephrol, 2000 May, 53(5), 319 - 24
Elevated tissue factor circulating levels in children with hemolytic uremic syndrome caused by verotoxin-producing E . coli; Kamitsuji H et al.; BACKGROUND: Microvascular thrombosis in the kidney plays an important role in the pathogenesis of hemolytic uremic syndrome (HUS) . Tissue factor (TF), present on the vascular surface of endothelial cells, binds factor VIIa . The complex initiates the coagulating cascade by activating factors X and IX . PATIENTS AND METHODS: In cases of HUS associated with verotoxin-producing E . coli (VTEC) infection, VTEC gastroenteritis without HUS and normal controls, we measured plasma concentrations of TF and tissue factor pathway inhibitor (TFPI) to evaluate their clinical significance . In children with non-HUS chronic renal failure (CRF), the TF levels were also measured as another control group . RESULTS: In the acute phase of HUS, plasma levels of TF and TFPI were significantly elevated, then returned to normal range in the recovery phase . The TF levels were closely correlated with the thrombin antithrombin-III complex, a marker of thrombin activity in circulating blood, and with creatinine clearance (Ccr) . Furthermore, a positive correlation was noted between plasma TF levels and plasma soluble thrombomodulin (sTM) levels, which is a marker of endothelial cell injury . The influence of decreased excretion from damaged kidneys should be considered since a definite lot correlation was observed between plasma TF levels and Ccr in children with non-HUS CRF . CONCLUSION: From these findings, we concluded that elevated TF circulating levels may also play an important role in blood-clotting activation observed in VTEC-HUS patients, and may also be a useful marker for renal damage.

Pediatr Infect Dis J, 2001 Mar, 20(3), 316 - 8
Hemolytic-uremic syndrome surveillance to monitor trends in infection with Escherichia coli O157 and non-O157 enterohemorrhagic E . coli in Austria; Fischer H et al.; Austrian data underline that relying on the number of enterohemorrhagic Escherichia coli (EHEC) O157 strains isolated from clinical specimens does not allow assessment of the actual incidence of EHEC infections . A hospital-based system for identification of hemolytic-uremic syndrome cases based on voluntary cooperation was established in 1995 and provides information needed to monitor trends in the incidence of O157 and non-O157 EHEC infections.

Fresenius J Anal Chem, 2001 Feb, 369(3-4), 295 - 301
Detection of E . coli using a microfluidics-based antibody biochip detection system; Stokes DL et al.; This work demonstrates the detection of E . coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays . The biochip features a 4 x 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform . The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors . The independently operating photodiodes allow simultaneous monitoring of multiple samples . In this study the sampling platform is a cellulosic membrane that is exposed to E . coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe . The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection . Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E . coli organisms . Selective detection of E . coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays.

Yonsei Med J, 2001 Feb, 42(1), 114 - 9
Increase in rat plasma antioxidant activity after E . coli lipopolysaccharide administration; Lee KY et al.; It is well recognized that the sensitivity of animals to lipopolysaccharide (LPS) endotoxin varies tremendously . And, it has been recently observed that Sprague-Dawley rats dramatically increase the activity of hepatic endogenous antioxidative enzyme systems after LPS administration . This finding suggests that the relative resistance of rats to LPS may be related to a concomitant increase in the activities of the hepatic antioxidant systems . This study was designed to examine if the above reported hepatic change in rats given LPS could be observed at the systemic level . Male Sprague-Dawley or Wistar rats, weighing 250 - 350 g, were given increasing doses (10 - 100 mg/kg) of LPS i.p . under 1.0% isoflurane anesthesia . Antioxidant capacity (AOC), blood gas analysis, and the cardiovascular parameters of the arterial blood of animals were determined over a 4 hour period following LPS administration . In addition, we studied the effect of pretreatment with the non-specific nitric oxide synthase inhibitor, L-N(G)-Nitroarginine methyl ester hydrochloride (L-NAME), given 50 mg/kg s.c . one and 24 hours before the administration of 20 mg/kg LPS i.p . in Sprague-Dawley rats . Rats given sufficiently high doses of E . coli LPS to produce behavioral effects also showed increased plasma AOCs in the early period after the administration of LPS . Similar changes were noted in Sprague-Dawley and Wistar rat strains, but at different doses that reflect their differential sensitivities to the LPS induced inflammatory response . Also, the resistance of the Sprague-Dawley strain of rats to LPS was not altered by the prior administration of L-NAME, nor was the plasma AOC altered . In conclusion, our study suggests that the rat strains are relatively resistant to develop the toxic signs of LPS in the early period after the administration of LPS, especially in Sprague-Dawley rats . Moreover, endotoxin-induced increases in plasma AOC may contribute to the rats' resistance to LPS intoxication.

J Immunol Methods, 2001 May 1, 251(1-2), 187 - 93
Protein affinity maturation in vivo using E . coli mutator cells; Coia G et al.; This protocol provides a simple in vivo strategy for introducing random mutations to a target DNA sequences using E . coli mutator cells . The method has been used in our laboratory for affinity maturation of proteins encoded by target DNA sequences . Selection conditions can be modified for antibody fragments with increased production levels . Growth conditions in E . coli mutator cells can be adjusted to introduce a single random point mutation per kilobase of DNA, approximately equivalent to one codon change per scFv fragment.

Biochemistry, 2001 Apr 10, 40(14), 4261 - 71
Structural basis for the functional switch of the E . coli Ada protein; Lin Y et al.; The Escherichia coli protein Ada specifically repairs the S(p) diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center . The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes . Ada thus acts as a chemosensor to activate repair mechanisms in situations of methylation damage . Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer . To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated . Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada . The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA . This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group.

Virology, 2001 Mar 15, 281(2), 272 - 80
A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E . coli; Yamshchikov V et al.; Infectious clone methodology is a valuable tool of modern experimental virology . However, its use is often constrained by the instability of infectious clone constructs during propagation in E . coli . To circumvent this problem, we have devised a strategy that could be suitable for design of +RNA virus molecular clones in general . An infectious clone is assembled as "infectious DNA," and expression of problem regions present in the viral cDNA is prevented during propagation in E . coli by insertion of short introns . To demonstrate the feasibility of this approach, a highly unstable Japanese encephalitis flavivirus infectious clone has been successfully converted into a remarkably stable infectious DNA construct with the specific infectivity of 10(6) pfu/microg in cell culture . The proposed strategy may be useful in the design of self-amplifying gene therapy vectors and development of new immunization methodologies, and could facilitate creation of molecular repositories of existing viral vaccines .

Genes Dev, 2001 Mar 15, 15(6), 748 - 61
Topoisomerase IV, alone, unknots DNA in E . coli; Deibler RW et al.; Knotted DNA has potentially devastating effects on cells . By using two site-specific recombination systems, we tied all biologically significant simple DNA knots in Escherichia coli . When topoisomerase IV activity was blocked, either with a drug or in a temperature-sensitive mutant, the knotted recombination intermediates accumulated whether or not gyrase was active . In contrast to its decatenation activity, which is strongly affected by DNA supercoiling, topoisomerase IV unknotted DNA independently of supercoiling . This differential supercoiling effect held true regardless of the relative sizes of the catenanes and knots . Finally, topoisomerase IV unknotted DNA equally well when DNA replication was blocked with hydroxyurea . We conclude that topoisomerase IV, not gyrase, unknots DNA and that it is able to access DNA in the cell freely . With these results, it is now possible to assign completely the topological roles of the topoisomerases in E . coli . It is clear that the topoisomerases in the cell have distinct and nonoverlapping roles . Consequently, our results suggest limitations in assigning a physiological function to a protein based upon sequence similarity or even upon in vitro biochemical activity.

Protein Sci, 2001 Jan, 10(1), 116 - 28
Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase from E . coli: fragment complementation and circular permutation reveal stable, alternatively folded forms; Smith VF et al.; The effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic and biochemical methods . Cleavage of the backbone after position 86 resulted in two fragments, (1--86) and (87--159) each of which are poorly structured and enzymatically inactive . When combined in a 1 : 1 molar ratio, however, the fragments formed a high-affinity (K(a) = 2.6 x 10(7) M(-1)) complex that displays a weakly cooperative urea-induced unfolding transition at micromolar concentrations . The retention of about 15% of the enzymatic activity of full-length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild-type counterpart . In contrast, a circularly permuted form with its N-terminus at position 86 has similar overall stability to full-length DHFR, about 50% of its activity, substantial secondary structure, altered side-chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild-type protein . After addition of ligand or the tight-binding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more native-like secondary and tertiary structures . These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein-ligand interactions in stabilizing the active site architecture of DHFR.

Biochemistry, 2001 Mar 13, 40(10), 3117 - 26
Determination of an optimal potential window for catalysis by E . coli dimethyl sulfoxide reductase and hypothesis on the role of Mo(V) in the reaction pathway; Heffron K et al.; Protein film voltammetry (PFV) of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC) adsorbed at a graphite electrode reveals that the catalytic activity of this complex Mo-pterin/Fe-S enzyme is optimized within a narrow window of electrode potential . The upper and lower limits of this window are determined from the potential dependences of catalytic activity in reducing and oxidizing directions; i.e., for reduction of DMSO (or trimethylamine-N-oxide) and oxidation of trimethylphosphine (PMe(3)) . At either limit, the catalytic activity drops despite the increase in driving force: as the potential is lowered below -200 mV (pH 7.0-8.9), the rate of reduction of DMSO decreases abruptly, while for PMe(3), an oxidative current is observed that vanishes as the potential is raised above +20 mV (pH 9.0) . Analysis of the waveshapes reveals that both activity thresholds result from one-electron redox reactions that arise, most likely, from groups within the enzyme; if so, they represent "switches" that reflect the catalytic mechanism and may be of physiological relevance . The potential window of activity coincides approximately with the appearance of the Mo(V) EPR signal observed in potentiometric titrations, suggesting that crucial stages of catalysis are facilitated while the active site is in the intermediate Mo(V) oxidation state.

Biochemistry, 2001 Mar 13, 40(10), 3047 - 55
The "catalytic" triad of isocitrate dehydrogenase kinase/phosphatase from E . coli and its relationship with that found in eukaryotic protein kinases; Oudot C et al.; The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E . coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue . As such, it belongs to the serine/threonine protein kinase family . However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure . In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases . In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403 . Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues) . Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases . Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification . Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain . In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme . However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme . On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected . Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.

Mol Biotechnol, 2000 Nov, 16(3), 253 - 60
Application of the E . coli trp promoter; Bass SH et al.; The Escherichia coli tryptophan (trp) promoter has been used extensively for the high level production of proteins on a small and large scale . This regulated promoter is readily available, relatively easy to turn on, and can be used in essentially any E . coli host background . This article gives a detailed use of the trp promoter including the design of expression vectors, subsequent culture conditions for promoter induction, and, finally, a protocol for the most common way of detecting the newly synthesized protein of interest . Its successful use for heterologous protein expression, however, sometimes requires consideration of parameters other than transcription such as translation initiation, translation elongation, and proteolysis . In this respect we offer guidance in getting through these post-transcriptional problems, which can occur with the use of any promoter.

Int J Food Microbiol, 2001 Feb 15, 63(3), 217 - 23
Characterization of Shiga toxin producing E . coli and O157 serotype E . coli isolated in France from healthy domestic cattle; Rogerie F et al.; A study was carried out in France in collaboration with the meat industry to investigate the occurrence and characteristics of Shiga toxin-producing E . coli (STEC) and O157 E . coli in a population of healthy bovines representative of French livestock . A total of 851 animals belonging to three bovine classes (106 young bulls, 374 dairy cows and 371 meat cows) were included in the study . Samples of feces and of the corresponding carcasses were collected from March 97 to August 97 in seven abattoirs spread throughout the national territory . STEC cultures from the 1702 samples were screened using PCR for the presence of stx genes . Positive samples were further subjected to colony blot hybridization and to O157-specific immunomagnetic separation . Probe-positive colonies and O157 colonies were then analyzed for the presence of virulence genes and phenotypic characters (serotype, Stx production) . In 154 (18.1%) feces and 91 (10.7%) carcass samples stx genes were detected . Two hundred and twenty-two STEC colonies were isolated from 67 (7.9%) feces and 16 (1.9%) carcass samples, with 183 STEC isolated from feces and 39 from carcasses . Only eight O157 isolates were collected from feces samples . None of these O157 E . coli isolates presented stx genes and thus could not be considered as pathogenic regarding hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) . In 3.2% of STEC isolated from feces and in 10.2% of STEC from carcasses eae genes were detected . In 17% of STEC from feces and in 30.7% from carcasses ehx genes were detected . Using these data, the 222 STEC colonies could be classified in 11 different 'virulence patterns' (presence/absence of stx1, stx2, eae and ehx genes), showing that more than 77% of isolates presented only one virulence factor . Only three STEC on 222 colonies (1.3%) presented the three virulence factors stx, eae and ehx in association, none of them reacting with antisera specific for enterohemorrhagic E . coli . (EHEC) . These data, together with the fact that only five isolates on the 222 (2.2%) reacted with such antisera (three O111 and two O26 isolates) demonstrated that the natural bacterial populations isolated during this study were clearly distinct from EHEC.

J Vet Diagn Invest, 2001 Jan, 13(1), 26 - 9
Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene and its relationship with fimbrial and enterotoxin genes in E . coli isolated from diarrheic piglets; Choi C et al.; A total of 720 Escherichia coli strains isolated from diarrheic piglets on 756 swine farms were screened for the presence of the enteroaggregative E . coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR) . Escherichia coli strains that carried EAST1 genes were also tested by PCR for the presence of 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa and STb), and 1 heat-labile enterotoxin (LT) gene . One hundred sixty-four (22.7%) of the 720 E . coli isolates carried genes for EAST1 . Of these 164 isolates, 62 (37.8%) carried EAST1 genes only, 11 (6.7%) carried genes for at least 1 of the fimbrial adhesins, 51 (31.1%) carried genes for at least 1 of the enterotoxins, and 40 (23.8%) carried genes for at least 1 of the fimbrial adhesins and enterotoxins . Forty-six percent of strains that carried EAST1 genes carried STa genes, and 16% of strains that carried EAST1 genes carried F4 . The isolation rate of enterotoxigenic E . coli strains carrying genes for EAST1 gene was 63% . The 6 major genotypes observed in this study (in decreasing order) were EAST1+, EAST1+STa+, EAST1+STa+STb+, EAST1+STa+F5+, EAST1+STa+F4+, and EAST1+STb+F4+ . EAST1 is widely prevalent among diarrheagenic strains of E . coli and may represent an important virulence determinant in the pathogenesis of enteric colibacillosis of preweaned pigs.

Chin Med Sci J, 1997 Mar, 12(1), 37 - 40
Expression of HBV Pre S1 peptide in E . coli and product characterization; Mi Z et al.; HBV Pre S1 sequence is supposed to play an important role in the infection of HBV . Presence of Pre S1/anti-Pre S1 in serum has valuable clinical implications . In order to improve the study of Pre S1, Pre S1 sequence was overexpressed in E . coli as a fusion protein with MBP (Maltose-binding protein), and anti-Pre S1 antiserum was elicited in rabbits by Pre S1-MBP purified by affinity chromatography . The recombinant plasmid constructed from pMAL-cRI expressed the 106aa Pre S1 sequence at the C terminal of MBP by tac promoter . The resulting protein is about 54 kD in size . Western-blot analysis confirmed its reactivity with antiserum derived from synthetic Pre S1 peptide and serum from patients with acute hepatitis B (AHB) . ELISA showed that Pre S1-MBP and Dane particles purified from AHB patient's serum reacted with antiserum against synthetic Pre S1 peptide, and this reaction was specifically inhibited by synthetic Pre S1 peptide . ELISA also demonstrated that antiserum against Pre S1-MBP reacted with synthetic Pre S1 peptide, but not with synthetic HCV peptide or HEV peptide.

J Microbiol Methods, 2001 Apr, 44(3), 225 - 33
Optimization of a fluorescent-based phosphor imaging dot blot DNA hybridization assay to assess E . coli virulence gene profiles; Zhang L et al.; To increase the efficiency and consistency in screening Escherichia coli for virulence genes, a Phosphor Imager was adopted for signal detection in Dot Blot DNA hybridization replacing X-ray film read by eye . We assessed not only the reliability of the instrument-based procedure, but the impact of going from an outcome measured by visualization on a semi-quantitative scale to a digitized readout on an interval scale . We analyzed technical and biological variability of the assay and the factors contributing to the variability . In spite of high variability both within and between membranes in the Phosphor Imager readings, we were able to define classification rules for gene presence that were remarkably consistent . Using the X-ray film signal detection procedure with Southern confirmation as a gold standard, we obtained a sensitivity and specificity of 87-99% for a rule requiring no retesting for all but one gene probe.

Genes Dev, 2001 Mar 1, 15(5), 491 - 506
Fine structure of E . coli RNA polymerase-promoter interactions: alpha subunit binding to the UP element minor groove; Ross W et al.; The alpha subunit of E . coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers . We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin . Our results suggest that alpha interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to alpha binding . On the basis of these and previous results, we propose a model in which alpha contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs . Furthermore, our experiments extend existing information about recognition of the core promoter by sigma(70) by identifying functional groups in the major grooves of the -35 and -10 hexamers in which modifications interfere with RNAP binding . These studies greatly improve the resolution of our picture of the promoter-RNAP interaction.

J Mol Biol, 2001 Feb 16, 306(2), 363 - 73
Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E . coli; Fribourg S et al.; Using the human basal transcription factors TFIID and TFIIH as examples, we show that pairwise coexpression of polypeptides in Escherichia coli can be used as a tool for the identification of specifically interacting subunits within multiprotein complexes . We find that coexpression of appropriate combinations generally leads to an increase in the solubility and stability of the polypeptides involved, which means that large amounts of the resulting complexes can immediately be obtained for subsequent biochemical and structural analysis . Furthermore, we demonstrate that the solubilization and/or the proper folding of a protein by its natural partner can be used as a monitor for deletion mapping to determine precise interaction domains . Coexpression can be used as an alternative or complementary approach to conventional techniques for interaction studies such as yeast two-hybrid analysis, GST pulldown and immunoprecipitation.

J Mol Biol, 2001 Feb 16, 306(2), 213 - 25
Interaction of the C-terminal domain of the E . coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface; Yasuno K et al.; The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA . Previously, we determined the solution structure of alphaCTD . Here, we investigated the interaction between alphaCTD and UP element DNA by NMR . DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA . Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding . There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay . We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending . Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA . The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction . Based on these observations, we propose a model for the UP element-alphaCTD complex.

Virus Res, 2001 Apr, 74(1-2), 59 - 73
Expression of deletion mutants of the hepatitis B virus protein HBx in E . coli and characterization of their RNA binding activities; Rui E et al.; The hepatitis B virus protein HBx has been implicated in the development of liver cancer . It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner . This DNA binding activity might be relevant for HBx oncogene character . To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E . coli . Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA . The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half . AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation . By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide . This new aspect of HBx function is discussed in the context of cellular transformation.

Bioorg Khim, 2000 Oct, 26(10), 728 - 34
{Cleavage of RNA in hybrid duplexes by ribonuclease H from E . coli . I . Substrate properties of complexes formed by RNA and tandem of short oligodeoxyribonucleotides}; Vorob'ev PE et al.; We studied the E . coli RNase H cleavage of a 5'-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20), whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide: (RNA/tandem) . It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3'-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5'-labeled fragments . On hydrolysis of RNA in complex RNA/tandem, the same products were registered, but their accumulation rates in this case differed . Thus, the initial rates of accumulation of the 17- and 8-mer were close . Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction, but only after a considerable lag period in complex RNA/p20 . These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation . This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem . Although the order of recognition and cleavage of RNA target by ribonuclease H depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness.

J Protein Chem, 2000 Jul, 19(5), 389 - 97
Characterization of glyoxalase I (E . coli)-inhibitor interactions by electrospray time-of-flight mass spectrometry and enzyme kinetic analysis; Stokvis E et al.; Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis . An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid . The E . coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I . Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I . Two specific binding sites were observed per dimer . The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-(2-{3-(hexyloxy)benzoyl}-vinyl)glutathione . Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction . This study shows that selective inhibitors may exist for the E . coli homologue of the glyoxalase I enzyme.

Bioorg Med Chem, 2001 Jan, 9(1), 51 - 6
Chemical synthesis and biological investigation of a 77-mer oligoribonucleotide with a sequence corresponding to E . coli tRNA(Asp); Persson T et al.; A 77-mer RNA with the sequence of Eschlerichia coli tRNA(Asp) has been chemically synthesised using standard automated phosphoramidite chemistry with the coupling reagent 4,5-dicyanoimidazole (DCI) . The synthesis was carried out on a 1000 A CPG-column and . after deprotection and gel purification, a yield of about 7 mmol with a purity of > 95% was reproducibly obtained . By comparing automated synthesis of the 77-mer RNA using 1H-tetrazole and DCI as activator, DCI is advantageous in producing longer RNAs . However, for shorter RNAs ( <40 mer) no difference could be observed . In addition to the all-ribo tRNA(Asp) carrying the wild-type sequence, two variants were synthesised, one with a single C to G48 mutation and the second with a 2'-deoxy modification at C48 . The three tRNAs were tested for their aminoacylation efficiency and high affinity binding to E . coli RNase P RNA . The results demonstrate that chemically synthesised 77-mer oligoribonucleotides can be successfully used for structure function studies.

Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 423 - 8
{Cloning and expression of the E . coli serine hydroxymethyltransferase gene (glyA)}; Shen T et al.; The E . coli K12 glyA gene(13 kb), encoding serine hydroxymethyltransferase (SHMT), has been cloned in the plasmid vector pBR329 using insertion inactivation and complementation test . Subcloning of segments of the original insert (13 kb) into plasmids pBR322, pBR329 and pSMY901 established that a 2.6 kb PstI-EcoR fragment carries the glyA gene . The 12 strains of transforments containing recombined plasmid . were obtained . SHMT and glyA gene product level in strains carrying glyA plasmids were different . No SHMT activity was observed in host strains . The glyA gene products for JM109(pSM13), K12(pSM13), K12(pSM14) and K12(pSM15) account for 15.7%, 15.4%, 11.8%, and 9.48% of the total dissoluble cell protein, respectively.

Am J Physiol Gastrointest Liver Physiol, 2001 Feb, 280(2), G216 - 21
Effect of E . coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice; Charney AN et al.; We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice . Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions . Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice . In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G) . Serosal STa had no effect . In distal colon neither mucosal nor serosal STa affected ion transport . In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon . In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice . We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa.

J Infect Dis, 2001 Mar 1, 183(5), 762 - 72 Epub 2001 Feb 08.
Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E . coli (EPEC) serogroups that carry EAE and lack the EPEC adherence factor and Shiga toxin DNA probe sequences; Vieira MA et al.; This study was conducted to characterize the virulence potential of 59 Escherichia coli strains carrying EAE and lacking the enteropathogenic E . coli adherence factor and Shiga toxin probe sequences . In hybridization studies, all strains carried the locus of enterocyte effacement (LEE)-associated DNA sequences . Of the other 15 virulence DNA sequences tested, HLY was the most frequent (44.1%); 17 combinations of these sequences were found, but strains carrying EAE only (EAE profile) were the most frequent (35.6%) . Except for 1 cytodetaching strain, all others adhered to HeLa and Caco-2 cells, most of which (approximately 75.0%) showed variations of the localized adherence pattern . Actin accumulation was detected in 75.9% of the nondetaching strains . Most strains had LEE, probably inserted in pheU (49.2%), and presented a nontypeable intimin (83.1%) . Translocated intimin receptor-derived DNA sequences correlated with enteropathogenic and enterohemorrhagic E . coli in 61.0% and 32.0% of the strains, respectively . Thirty-five different serotypes were found . Only strains with the EAE profile were associated with diarrhea (P=.039).

Bioelectromagnetics, 2001 Feb, 22(2), 79 - 86
Effect of static magnetic field on E . coli cells and individual rotations of ion-protein complexes; Binhi VN et al.; The effect of week static magnetic fields on Escherichia coli K12 AB1157 cells was studied by the method of anomalous viscosity time dependencies (AVTD) . The AVTD changes were found when E . coli cells were exposed to static fields within the range from 0 to 110 microT . The dependence of the effect on the magnetic flux density had several extrema . These results were compared with theoretical predictions of the ion interference mechanism . This mechanism links the dissociation probability of ion--protein complexes to parameters of magnetic fields . The mechanism was extended to the case of rotating complexes . Calculations were made for several ions of biological relevance . The results of simulations for Ca(2+), Mg(2+), and Zn(2+) showed a remarkable consistency with experimental data . An important condition for this consistency was that all complexes rotate with the same speed approximately 18 revolutions per second (rps) . This suggests that the rotation of the same carrier for all ion--protein complexes may be involved in the mechanism of response to the magnetic field . We believe that this carrier is DNA .

J Appl Microbiol, 2001 Feb, 90(2), 201 - 7
Growth and survival of E . coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk; Maher MM et al.; AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk . METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E . coli O157:H7, and enumeration was carried out using CT-SMAC . From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1 . During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days . The presence of E . coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR . CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E . coli O157:H7 to levels that permitted survival during ripening and extended storage . SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E . coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.

Genes Cells, 2000 Dec, 5(12), 953 - 63
Interaction of ribosome recycling factor and elongation factor EF-G with E . coli ribosomes studied by the surface plasmon resonance technique; Ishino T et al.; BACKGROUND: Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides . How RRF dissociates the complex has long been puzzling . Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does . RESULTS: The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique . RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions . This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro . Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro . The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G . CONCLUSIONS: These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action . The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.

Vet Immunol Immunopathol, 2000 Dec 29, 77(3-4), 221 - 32
Differential gene regulation of interleukin-1 ligands and receptors in bovine peripheral blood neutrophils and mononuclear cells in response to E . coli lipopolysaccharide (LPS); Beckman MJ et al.; The proinflammatory cytokine, interleukin-1, plays a prominent role in the inflammatory reactions that characterize numerous diseases . In this study, we examined the gene expression for bovine IL-1 ligands and receptors by bovine peripheral blood mononuclear cells (MNCs) and neutrophils (PMNs) in response to E . coli lipopolysaccharide (LPS) in vitro . Gene expression of mRNA for IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), type 1 IL-1 receptor, type 2 IL-1 receptor, and IL-1 beta converting enzyme (ICE), were measured by a semi-quantitative RT-PCR technique . LPS had little effect on type 1 IL-1R expression in MNC, whereas, it strongly up-regulated type 1 IL-1R expression in PMNs . Co-incubation of PMNs with LPS and bovine recombinant IL-1beta had little additional effect on type 1 IL-1R expression . Incubation of MNCs with LPS resulted in up-regulation of IL-1beta, IL-1ra, and type 2 IL-1R, no change in IL-1alpha, and a decrease in ICE gene expression . Incubation of PMNs with LPS up-regulated IL-1beta gene expression, whereas, IL-1alpha, IL-1ra, type 2 IL-1R and ICE were unchanged . This study provides evidence for differential regulation of gene products of the bovine IL-1 family by peripheral blood mononuclear cells (MNC) and neutrophils (PMNs) in response to E . coli LPS.

Free Radic Biol Med, 2001 Jan 1, 30(1), 51 - 61
Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E . coli: the role of selenocysteine in the catalytic activity; Bar-Noy S et al.; In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue . This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity . For expression of the human gene in E . coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E . coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene . Omission of this SECIS element from another construct resulted in termination at UGA . Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine . The three gene products were purified using a standard isolation protocol . Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar . The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction . With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.

Ann Surg Oncol, 2000 Dec, 7(10), 743 - 9
A novel approach for the identification of unique tumor vasculature binding peptides using an E . coli peptide display library; Brown CK et al.; BACKGROUND: Tumor neovascularization is necessary for continued tumor growth and metastasis . During the process of endothelial cell (EC) recruitment and tumor infiltration, specific molecular markers unique for this interaction are expressed on the EC surface . Targeting these molecular markers would, in effect, allow for specific tumor targeting . Tripeptide sequence motifs have previously been reported that will bind to angiogenic tumor ECs . These sequences were identified from in vivo phage peptide display libraries . The purpose of this study was to use a more simplified bacterial peptide display library in an in vitro system to seek out peptide motifs with unique binding to tumor microvasculature . METHODS: FliTrx is a bacterial peptide display library containing the entire repertoire of possible random dodecapeptides expressed on the flagella tip of E . coli . Two EC populations were used for the screening process, Matrigel invading cells (MAGIC) and tumor-derived endothelial cells (TDEC) . MAGIC are obtained from ECs that infiltrate a subcutaneous fibroblast growth factor-containing Matrigel deposit, and TDEC are ECs selectively obtained from tumor vasculature . FliTrx cells were incubated with MAGIC at 4 degrees C to remove any potential clones displaying peptides that will bind to nonspecific EC surface targets . The non-binding cells were then incubated with TDEC, allowing for clones displaying potential binding peptides to bind tumor specific targets on TDECs . The bacterial population was then expanded and this "panning" process was carried out a total of five times . Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs . RESULTS: Recurring peptide sequences were detected that were 3-mers (13 sequences) and 4-mers (4 sequences) . Of the 3-mers, four repeated 3 times, whereas none of the 4-mers repeated more than twice . All of the repeated sequences were basic in charge, and arginine was the most commonly seen amino acid . A tripeptide basic-basic-nonpolar amino acid arrangement was the most prevalent charge sequence in all repetitive motifs (17 repeat sequences) . Two test peptides showed TDEC binding specificity, and both conformed to the basic-basic-nonpolar motif . CONCLUSIONS: We report peptide sequences derived from panning an in vitro system designed to detect tumor-EC specific markers . These putative motifs may serve as molecular determinants for a novel therapeutic modality aimed at specifically targeting tumors through tumor angiogenic vessels.

J Mol Biol, 2000 Dec 15, 304(5), 765 - 78
Interaction of the E . coli DNA G:T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence; Turner DP et al.; The Escherichia coli vsr endonuclease recognises G:T base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T . The enzyme shows a preference for G:T mismatches within a particular sequence context, derived from the recognition site of the E . coli dcm DNA-methyltransferase (CC{A/T}GG) . Thus, the preferred substrate for the vsr protein is (CT{A/T}GG), where the underlined T is opposed by a dG base . This paper provides quantitative data for the interaction of the vsr protein with a number of oligonucleotides containing G:T mismatches . Evaluation of specificity constant (k(st)/K(D); k(st)=rate constant for single turnover, K(D)=equilibrium dissociation constant) confirms vsr's preference for a G:T mismatch within a hemi-methylated dcm sequence, i.e . the best substrate is a duplex (both strands written in the 5'-3' orientation) composed of CT{A/T}GG and C(5Me)C{T/A}GG . Conversion of the mispaired T (underlined) to dU or the d(5Me)C to dC gave poorer substrates . No interaction was observed with oligonucleotides that lacked a G:T mismatch or did not possess a dcm sequence . An analysis of the fraction of active protein, by "reverse-titration" (i.e . adding increasing amounts of DNA to a fixed amount of protein followed by gel-mobility shift analysis) showed that less than 1% of the vsr endonuclease was able to bind to the substrate . This was confirmed using "competitive titrations" (where competitor oligonucleotides are used to displace a (32)P-labelled nucleic acid from the vsr protein) and burst kinetic analysis . This result is discussed in the light of previous in vitro and in vivo data which indicate that the MutL protein may be needed for full vsr activity .

Biochemistry, 2000 Dec 26, 39(51), 16244 - 51
Identification of active site residues in E . coli ketopantoate reductase by mutagenesis and chemical rescue; Zheng R et al.; Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate . The pH dependence of V and V/K for the E . coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism . To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256 . Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in V(max), and 336- and 63-fold increases in the K(m) value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties . The V(max) for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively . The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume . The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction . The rescue efficiencies for the K176A mutant by primary amines were independent of the pK(a) value of the rescue agents (Bronsted coefficient, alpha = -0.004 +/-0.008) . Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (k(cat)/K(m) = 2x10(6) M(-1) s(-1) and (b) the small primary deuterium kinetic isotope effects, (D)V = 1.3 and (D)V/K = 1.5, observed for the wild-type enzyme . Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant ((D)V = 3.0, (D)V/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased . The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding . The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity . These results indicate that Lys176 and Glu256 of the E . coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.

Genes Cells, 2000 Nov, 5(11), 873 - 884
Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E . coli in vitro; Taghbalout A et al.; BACKGROUND: Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein . SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro . SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo . RESULTS: We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC . (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect . The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region . CONCLUSIONS: SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.

Virology, 2000 Dec 20, 278(2), 578 - 86
Recombinant hepatitis delta antigen from E . coli promotes hepatitis delta virus RNA replication only from the genomic strand but not the antigenomic strand; Sheu GT et al.; Hepatitis delta antigen (HDAg) of hepatitis delta virus (HDV) typically consists of two related protein species . The small HDAg (S-HDAg) is a 24-kDa protein of 195 amino acids and the large HDAg (L-HDAg) is a 27-kDa protein with an additional 19 amino acids at its C-terminus . These two proteins have distinct functions in the HDV life cycle . We have developed conditions for expressing S-HDAg and L-HDAg in E . coli as soluble proteins to facilitate large-scale purification . These proteins were purified to homogeneity and shown to be biologically active . Transfection of the purified recombinant S-HDAg together with HDV genomic RNA resulted in viral RNA replication . Surprisingly, the purified S-HDAg could not initiate replication from the antigenomic-sense HDV RNA, even though the latter led to RNA replication when transfected with an mRNA encoding the S-HDAg . These results suggest that initiation of HDV RNA synthesis from the antigenomic RNA may require a form of HDAg that is modified in mammalian cells; in contrast, RNA synthesis from the genomic RNA could be initiated by the recombinant S-HDAg from E . coli . Interestingly, the purified L-HDAg appeared as multiple protein species, including one corresponding to S-HDAg, probably as a result of degradation . The partially proteolyzed L-HDAg also initiated HDV RNA replication under the same conditions . These results add to the mounting evidence that genomic- and antigenomic-strand HDV RNA syntheses are carried out by different mechanisms .

J Mol Biol, 2001 Jan 5, 305(1), 167 - 77
Localization of the protein L2 in the 50 S subunit and the 70 S E . coli ribosome; Willumeit R et al.; The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex . The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation . L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit . The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix . The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs .

Neuroradiology, 2000 Oct, 42(10), 778 - 80
De novo development of presumed cavernomas following resolution of E . Coli subdural empyemas; Fender LJ et al.; Cavernomas fall within the group of angiographically occult lesions and may be found in up to 4 % of the population {1} . They may occur at any age, and with the advent of MRI incidental cavernomas are increasingly identified . The pathogenesis is uncertain . Familial cases are well recognised with a reported prevalence of 10-15 % {2-3} . The incidence of new lesions has been reported at 0.4 lesions per patient per year in cases with familial cavernomas {4} . Presumed cavernomas have been documented following radiation for malignancy {5-6}, and stereotactic cerebral biopsy {7} . There have been no previously documented cases of de novo genesis of cavernomas following bacterial meningitis and subdural empyemas.

Eur J Biochem, 2000 Dec, 267(24), 7015 - 23
Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli . Structural and functional properties of wild-type, mutants and E . coli enzymes; Incharoensakdi A et al.; Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline . Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes . To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed . These enzymes together with Escherichia coli BADH were expressed in E . coli and purified . The affinities for betaine aldehyde were similar in the spinach and E . coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E . coli BADH . A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E . coli BADH, showed properties which resembled spinach BADH more than E . coli BADH . The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes . All spinach BADHs were dimers whereas E . coli BADH was a tetramer . E . coli BADH was more stable at high temperature than spinach BADHs . The E103Q mutant was most labile to high temperature . These properties are discussed in relation to the structure of spinach BADH.

Eur Respir J, 2000 Oct, 16(4), 697 - 703
Modulation by pentobarbital of neutrophil responses to inhaled E . coli endotoxin in sheep: role of lung epithelium; Peterson BT et al.; Neutrophils (PMNs) are implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) . The role of the epithelium in the modulation of PMN migration within the lungs was examined . Epithelial integrity and PMN concentrations in the lung air spaces and lymph were measured in sheep anaesthetized with either halothane (1-2.5%) or intravenous pentobarbital (12+/-4 mg x kg(-1) x h(-1)) . Ventilation with an aerosol containing 25 mg Escherichia Coli endotoxin (lipopolysaccharide; LPS) effected neutrophil recruitment to the air spaces . Lymphatic clearance of aerosolized 99mTc-DTPA provided an index of epithelial integrity . Three hours after the deposition of LPS, the lung lining fluid of sheep anaesthetized with halothane (n=7) had 4.9+/-3.2x10(6) PMN.mL(-1), but the lung lymph had almost no PMNs (3+/-8%) . Sheep anaesthetized with pentobarbital (n=6) had fewer PMNs in the air spaces (2.4+/-1.2X10(6) mL(-1)) and more PMNs in the lung lymph (30+/-20%) . Control sheep (n=5) that received no LPS had almost no PMNs in the airspaces or lung lymph, regardless of the anaesthesia . Three additional sheep that remained awake after receiving LPS also had no PMNs in the lung lymph . The PMN fraction in the lung lymph correlated well with the extra-alveolar epithelial permeability measured by lymphatic clearance of aerosolized diethylenetriamine penta-acetic acid (r=0.81, p<0.001) . Aerosolized lipopolysaccharide recruits neutrophils into the lungs of sheep, but they appear to remain in the airspaces unless extra-alveolar permeability is increased by agents such as pentobarbital.

Schweiz Arch Tierheilkd, 2000 Nov, 142(11), 625 - 30
{Main virulence factors in Escherichia coli isolates from swine over two weeks old with edema disease and/or E . coli diarrhea}; Sarrazin E et al.; The OK antigens and the fimbriae F4 of E . coli with haemolysis isolated from 113 cases of oedema disease and/or diarrhoea were identified serologically . The genes for F18 and for enterotoxins LT, STIa and STII as well as Shigatoxin Stx2e were determined by PCR . Fimbrial variants F18ab and F18ac were distinguished by means of indirect immunofluorescence on smears prepared from the intestinal mucosa and from cultures grown under appropriate conditions . Adhesive fimbriae were detected with every case or isolate, respectively, by means of at least one out of the techniques mentioned above . The serogroup O149:K91 with fimbriae F4ac (K88ac) and genes for the enterotoxins LT and STII was most prevalent . Serogroup O139:K12 with fimbriae F18ab and the gene for Stx2e was second, whereas serogroups O141ab and O141ac with fimbriae F18ac and genes for Stx2e, STII and often LT were much less prevalent . The serogroup O147:K89 with fimbriae F18ac, and genes for STIa and STII was detected for the first time in Switzerland.

Biotechnol Prog, 2000 Nov-Dec, 16(6), 917 - 21
Nitrite inhibition of Vitreoscilla hemoglobin (VHb) in recombinant E . coli: direct evidence that VHb enhances recombinant protein production; Aydin S et al.; Bacteria engineered with the gene (vgb) encoding Vitreoscilla hemoglobin (VHb) typically produce more protein than unengineered cells, and it has generally been assumed that VHb is responsible for this effect . Here, using matched strains of E . coli that bear a recombinant alpha-amylase gene (MK57) or the alpha-amylase gene and vgb (MK79), we provide evidence supporting this assumption . Sodium nitrite (which is known to inhibit heme proteins) was tested over a range of concentrations regarding effects on growth, alpha-amylase production, respiration, and VHb function in MK57 and MK79 . Nitrite concentrations were identified at which respiration of cell membranes was inhibited only slightly and to approximately equal degrees in both strains, while whole cell respiration was inhibited to a greater extent and about twice as much in MK79 as MK57 . This suggests that these concentrations inhibit VHb while having a much smaller effect on cytochrome oxidase . Direct measurements of VHb showed, in fact, that the same nitrite concentrations greatly decreased the levels of active (ferrous) and, to a somewhat lesser extent, total (ferrous plus ferric) VHb in MK79 . Finally, these same nitrite concentrations reversed the advantage regarding alpha-amylase production of MK79 over MK57 seen at 0 mM nitrite, linking the presence of active VHb with the increase in alpha-amylase production.

Eur J Immunol, 2000 Dec, 30(12), 3522 - 32
Recombinant human single-chain MHC-peptide complexes made from E . coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens; Denkberg G et al.; Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies . In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides . The scMHC molecule was composed of beta2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker . Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1 . The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides . We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-gamma, and were able to stain specifically CTL lines . Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Genes Dev, 2000 Nov 15, 14(22), 2881 - 92
Preferential relaxation of positively supercoiled DNA by E . coli topoisomerase IV in single-molecule and ensemble measurements; Crisona NJ et al.; We show that positively supercoiled {(+) SC} DNA is the preferred substrate for Escherichia coli topoisomerase IV (topo IV) . We measured topo IV relaxation of (-) and (+) supercoils in real time on single, tethered DNA molecules to complement ensemble experiments . We find that the preference for (+) SC DNA is complete at low enzyme concentration . Otherwise, topo IV relaxed (+) supercoils at a 20-fold faster rate than (-) supercoils, due primarily to about a 10-fold increase in processivity with (+) SC DNA . The preferential cleavage of (+) SC DNA in a competition experiment showed that substrate discrimination can take place prior to strand passage in the presence or absence of ATP . We propose that topo IV discriminates between (-) and (+) supercoiled DNA by recognition of the geometry of (+) SC DNA . Our results explain how topo IV can rapidly remove (+) supercoils to support DNA replication without relaxing the essential (-) supercoils of the chromosome . They also show that the rate of supercoil relaxation by topo IV is several orders of magnitude faster than hitherto appreciated, so that a single enzyme may suffice at each replication fork.

Protein Expr Purif, 2000 Dec, 20(3), 451 - 61
The full-length, cytoplasmic C-terminus of the beta 2-adrenergic receptor expressed in E . coli acts as a substrate for phosphorylation by protein kinase A, insulin receptor tyrosine kinase, GRK2, but not protein kinase C and suppresses desensitization when expressed in vivo; Doronin S et al.; The ability of the cytoplasmic, full-length C-terminus of the beta 2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested . BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation . The mobility of BAC1 on SDS-PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds . Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E . coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors . Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry . Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK . The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character . The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1 . The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase . GRK2 catalyzed modest phosphorylation of BAC1 . Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the "activated" conformer required by GRKs is low . BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred . The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells . BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells . Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E . coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization .

Bioconjug Chem, 2000 Nov-Dec, 11(6), 777 - 88
Design and synthesis of novel glycopolythiophene assemblies for colorimetric detection of influenza virus and E . coli; Baek MG et al.; A novel family of glycopolythiophenes containing sialic acid or mannose ligands were prepared and evaluated for their ability to bind lectins, virus, and bacteria . For the set of glycopolythiophenes studied, the spacer-length between the polymer backbone and the ligand was varied to optimize binding interactions . The glycopolymers were blue-shifted (absorbance of ca . 400 nm) relative to the corresponding homo-polythiophenes (absorbance ca . 440 nm), suggesting a twisted conformation for the glycopolymers . The altered conformation is likely due to electrostatic or H-bonding interactions between the polymer chains, arising from the carbohydrate ligand . Further conformational changes in the polythiophene backbone were detected by the binding of specific receptors; lectins (wheat germ agglutinin, concanavalin A), Influenza virus, and Escherichia coli . The binding interactions result in an unusual red-shift in the visible absorption of the polymer backbone, suggesting a lengthening of the effective conjugated length upon interaction of the ligand with its congnate receptor . These conjugated glycopolymeric systems offer a potentially new platform for the detection of molecular binding interactions.

Gene Ther, 2000 Oct, 7(20), 1738 - 43
In vivo sensitization of ovarian tumors to chemotherapy by expression of E . coli purine nucleoside phosphorylase in a small fraction of cells; Gadi VK et al.; This report examines a major barrier to suicide gene therapy in cancer and other diseases: namely, bystander cell killing . Existing vectors for in vivo gene delivery are inefficient and often transduce or transfect less than 1% of target cells . The E . coli PNP gene brings about cellular necrosis under conditions when 1 in 100 to 1 in 1000 cells express the gene product in vitro . In vivo bystander killing at or near this magnitude has not been reported previously . In the present experiments, transfection of cells with the E . coli PNP gene controlled by a SV40 promoter resulted in 30 nmol 6-methyl purine deoxyriboside (MeP-dR) converted per milligram tumor cell extract per hour (or conversion units (CU)) . This level of expression led to elimination of entire populations of tumor cells in vitro after treatment with MeP-dR . Much earlier killing was observed using a tat transactivated E . coli PNP vector (approximately seven-fold higher activity, 230 CU) . In vivo effects on tumor growth were next examined . Human ovarian tumors transfected with E . coli PNP were excised 5 days after i.p . implantation from the peritoneal cavities of mice in order to determine both E . coli PNP enzymatic activity and the fraction of cells expressing the gene . PNP activity at 5 days after gene transfer was approximately 170 CU and was expressed in approximately 0.1% of the tumor cells as judged by in situ hybridization . The expression of E . coli PNP at this level produced a 30% increase in life span (P < 0.001) and 49% reduction in tumor size (P < 0.005) after MeP-dR treatment, as compared with control tumors . Our observations lead to the conclusion that pronounced bystander killing by E . coli PNP is conferred in vivo, and that vectors capable of transgene expression in as few as one in 1000 cells can produce substantial antitumor effects if expression on a per cell basis is very high.

Int J Food Microbiol, 2000 Nov 1, 61(2-3), 127 - 36
Recovery of low-temperature stressed E . coli O157:H7 and its susceptibility to crystal violet, bile salt, sodium chloride and ethanol; Chou CC et al.; This study was conducted to investigate the alteration of some characteristics of E . coli O157:H7 subjected to various periods of storage at -5, -18 and -28 degrees C . Results revealed that the low-temperature treatments increased the susceptibility of E . coli O157:H7 to crystal violet, bile salt, sodium chloride and ethanol . In general, the susceptibility of E . coli O157:H7 subjected to storage at -18 degrees C increased most significantly . The susceptibility of E . coli O157:H7 to the tested agents increased as the period of low-temperature storage extended, regardless of storage temperature . Among the various nitrogen and carbon sources tested, tryptone and soytone were the most effective nitrogen sources, while glucose and maltose were the most effective carbon sources for the growth of the low-temperature stressed cells . When growing the stressed E . coli O157:H7 in media containing the same nitrogen source or carbon source, their lag period increased as the time of frozen storage increased . It was also noted that in general, the recovery of the low-temperature stressed E . coli O157:H7 was highest on tryptic soy agar followed by Modified eosin methylene blue agar, while recovery on MaConkey sorbitol agar and Modified MaConkey sorbitol agar was lowest.

Biochemistry, 2000 Nov 14, 39(45), 13856 - 61
Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E . coli undecaprenyl pyrophosphate synthase catalysis; Pan JJ et al.; Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate . We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis . The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity . Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs . Its IPP K(m) value has only minor change . The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat) . The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type . These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding . The E213 residue in region V is also important in IPP binding as well as catalysis . Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A . Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs.

J Hepatol, 2000 Oct, 33(4), 528 - 36
Antibodies against the COOH-terminal region of E . coli ClpP protease in patients with primary biliary cirrhosis; Mayo I et al.; BACKGROUND/AIMS: The presence of antibodies in sera from patients with autoimmune diseases is an important tool for diagnosis and for providing insights into the mechanisms leading to autoimmunity . The aim of this study was to characterize new reactive antigens in liver autoimmune diseases . METHODS: Sera of patients with liver-related autoimmune (n=74) and non-liver-related autoimmune (n= 211) diseases, non-autoimmune liver diseases (n=18) and healthy controls (n=160) were evaluated for antibodies against E . coli ClpP protease (EClpP) and 20S proteasome by immunoblot analysis . RESULTS: Antibodies against EClpP were detected in 15 of 50 patients with primary biliary cirrhosis, in only one of 100 patients with systemic lupus erythematosus, and in three healthy subjects (Chi-square 59.1, d.f . 2, p< 0.001) . Antibodies to 20S proteasome were found in only 35 of 100 patients with systemic lupus erythematosus . All other sera from patients with autoimmune diseases, liver diseases other than primary biliary cirrhosis, and healthy controls were negative for both antigens . Both IgG and IgM classes of antibodies against EClpP were present in primary biliary cirrhosis patient sera with titers of 1/400-1/1000 . By using recombinant techniques and peptide ELISA, the immunodominant EClpP epitope recognized by the sera from primary biliary cirrhosis patients was localized in the amino acid sequences 177-194 (QIERDTERDRFLSAPEAV) within the COOH-terminal of EClpP . Affinity-purification of these anti-EClpP antibodies and immunoabsorption experiments established that the antibodies are specific for the bacterial EClpP . CONCLUSIONS: Bacterial ECIpP has been identified as a new antigen specifically reacting with sera from approximately one third of patients with primary biliary cirrhosis.

Appl Environ Microbiol, 2000 Nov, 66(11), 4926 - 34
Persistent colonization of sheep by Escherichia coli O157:H7 and other E . coli pathotypes; Cornick NA et al.; Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans . Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown . We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E . coli O157:H7 to that by other pathotypes of E . coli . Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E . coli O157:H7, two strains of enterotoxigenic E . coli (ETEC), and one strain of enteropathogenic E . coli . Both STEC strains and ETEC 2041 were given at either 10(7) or 10(10) CFU/strain/animal . The other strains were given only at 10(10) CFU/strain . We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization . However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes . The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10(7) or 10(10) CFU . One of the ETEC strains also persisted when inoculated at 10(10) CFU . However, in contrast to the STEC strains, it did not persist when inoculated at 10(7) CFU . These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E . coli.

Gene, 2000 Oct 17, 257(1), 87 - 97
Phylogenetic analysis of teneurin genes and comparison to the rearrangement hot spot elements of E . coli; Minet AD et al.; Teneurins are a novel family of transmembrane proteins conserved between invertebrates and vertebrates . There are two members in Drosophila, one in C . elegans and four members in mouse . Here, we describe the analysis of the genomic structure of the human teneurin-1 gene . The entire human teneurin-1 (TEN1) gene is contained in eight PAC clones representing part of the chromosomal locus Xq25 . Interestingly, many X-linked mental retardation syndromes (XLMR) and non-specific mental retardation (MRX) are mapped to this region . The location of the human TEN1 together with the neuronal expression makes TEN1 a candidate gene for XLMR and MRX . We also identified large parts of the human teneurin-2 sequence on chromosome 5 and sections of human teneurin-4 at chromosomal position 11q14 . Database searches resulted in the identification of ESTs encoding parts of all four human members of the teneurin family . Analysis of the genomic organization of the Drosophila ten-a gene revealed the presence of exons encoding a long form of ten-a, which can be aligned with all other teneurins known . Sequence comparison and phylogenetic trees of teneurins show that insects and vertebrates diverged before the teneurin ancestor was duplicated independently in the two phyla . This is supported by the presence of conserved intron positions between teneurin genes of man, Drosophila and C . elegans . It is therefore not possible to class any of the vertebrate teneurins with either Drosophila Ten-a or Ten-m . The C-terminal part of all teneurins harbours 26 repetitive sequence motifs termed YD-repeats . YD-repeats are most similar to the repeats encoded by the core of the rearrangement hot spot (rhs) elements of Escherichia coli . This makes the teneurin ancestor a candidate gene for the source of the rhs core acquired by horizontal gene transfer.

J Dairy Sci, 2000 Oct, 83(10), 2276 - 81
A comparison of two commercially available Escherichia coli J5 vaccines against E . coli intramammary challenge; Tomita GM et al.; The efficacy of two commercially available Escherichia coli J5 bacterins was investigated . Jersey cows were randomly assigned to one of three treatment groups: 1) unvaccinated controls, 2) vaccinated with J.VAC (Merial Limited, Athens, GA), and 3) vaccinated with J5 bacterin . All cows were vaccinated at drying off and at 2 wk before anticipated calving . Cows that were vaccinated with the J5 bacterin also received a third immunization at calving . One quarter of each cow was challenged with approximately 64 cfu of E . coli at 14 to 30 d postcalving . Immunization by either vaccine did not influence the severity of coliform mastitis; however, the mean number of colony-forming units of E . coli recovered from challenged quarters was significantly lower for immunized cows than for control cows at 144 h postchallenge . Serum and mammary secretion immunoglobulin (Ig)G, IgG1, and IgG2 titers against E . coli J5 whole-cell antigens were enhanced in vaccinated cows . Serum and mammary secretion IgM were not different among treatment groups . Somatic cell counts in milk from challenged quarters, rectal temperatures, and the clinical status of cows following intramammary challenge were not different among treatment groups.

Shi Yan Sheng Wu Xue Bao, 1997 Sep, 30(3), 285 - 92
{Cloning of variable region genes of anti-tetanus toxoid antibody and their expression as three kinds of engineered antibodies in E . coli}; Zan H et al.; The heavy and light chain variable region genes of anti-tetanus toxoid (TT) antibody and the heavy chain Fd genes were amplified and cloned through RT-PCR from mouse hybridoma cells . The sequences of VH and VK were determined . Fd gene fragments were expressed in E . coli . The ELISA results indicated that the expressed Fd showed antigen binding activity but was nonspecific . Furthermore, through SOE and PCR techniques, the VH and VK gene fragments together with ScFv linker were assembled into single chain antibody (ScFv) gene fragment . While together with human heavy chain CH 1 gene fragment and Fab linker, they were assembled into chimeric Fab gene fragment . The two assembled gene fragments were separately inserted into phagemid pHEN 1, which was a fd-based vector containing gene 3 encoding the minor coat protein . In presence of helper phage M 13-VCS the anti-TT phage-ScFv or phage-Fab were displayed on the surface of phage particles respectively . Results from phage-ELISA indicated that both phage antibodies were TT-specific.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 293 - 8
Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E . coli and chain-like adherent E . coli isolated from children with and without diarrhoea, in Londrina, Brazil; Gioppo NM et al.; A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells . Localised adherence was found only in isolates from cases . Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls . The AA isolates were tested for gene sequences associated with enteroaggregative E . coli (EAEC) . Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively . The aggA gene was not found, although 7.9% were positive for aggC . The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively . The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.

Radiats Biol Radioecol, 2000 Jul-Aug, 40(4), 378 - 81
{The effect of am umuC mutation on the induction of an SOS response in E . coli cella under the action of UV and gamma irradiation}; Komova OV et al.; The kinetics of the SOS induction in E . coli cells of wild type and deficient in umuC gene exposed to UV and gamma-rays were analysed . In the presence of UmuC protein SOS induction was 3-5.5 times lower and delayed for about 30 minutes after both UV and gamma rays . It was shown that decrease of the SOS induction in wild type cells irradiated by UV was due to more effective elimination of the photolesions from DNA by excision repair system . UmuCD-dependent inhibition of DNA replication was discussed as a possible mechanism allowing additional time for error-free repair.

Biochemistry, 2000 Oct 10, 39(40), 12303 - 11
Secondary structure and oligomerization of the E . coli glycerol facilitator; Manley DM et al.; The Major Intrinsic Proteins are found throughout the bacterial, plant, and animal kingdoms and are responsible for the rapid transport of water and other small, polar solutes across membranes . The superfamily includes the aquaporins, the aquaglyceroporins, and the glycerol facilitators . We have overexpressed and purified the Escherichia coli inner membrane glycerol facilitator . Approximately 7.5 mg of 95% pure protein is obtained from 1 L of Escherichia coli cells using immobilized metal affinity chromatography . Well-resolved matrix-assisted laser desorption ionization mass spectra were obtained by solubilization of the protein in octyl-beta-D-glucopyranoside (M(r) = 33 650.3; error approximately 0.4%) . The recombinant glycerol facilitator is inserted into the bacterial inner membrane, is functional, and is inhibited by HgCl(2) . Polyacrylamide gel electrophoresis suggests that the facilitator is predominantly monomeric when solubilized with dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, and sodium dodecyl sulfate, but that it self-associates, forming soluble oligomers when urea is used during extraction . Similar oligomeric species are demonstrated to exist in the bacterial membrane by chemical cross-linking experiments . Circular dichroism analysis shows that the protein is predominantly alpha-helical . Helix content is significantly higher in protein prepared in the absence of urea (42-55%) than in its presence (32%) . A possible role for the facilitator oligomers in interactions with, and regulation of, the glycerol kinase is discussed.

Biochemistry, 2000 Oct 10, 39(40), 12274 - 83
Sequence determinants for the recognition of the fork junction DNA containing the -10 region of promoter DNA by E . coli RNA polymerase; Matlock DL et al.; It has been recently suggested that E . coli RNA polymerase can specifically recognize a fork junction DNA structure, suggesting a possible role for such interaction in promoter DNA melting {Guo, Y., and Gralla, J . D . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 11655-11660} . We have determined here quantitatively, using a site-specific binding assay, the effects of base substitutions within the conserved -10 hexamer in the context of a short fork junction DNA on binding to RNA polymerase . Adenine at position -11 and thymine at position -7 were found to be critical for sequence-specific recognition of the DNA . The identities of bases at positions -9 and -8 were found to be not important for the binding whereas replacement of bases at positions -12 and -10 had a mild negative effect on the binding affinity . It was found that for the binding of fork DNA to RNA polymerase, specific sequence recognition was more important than specific recognition of fork junction DNA structure . The pattern of relative importance of bases in the -10 region for binding RNA polymerase was generally consistent with the sequence conservation pattern observed in nature where positions -11 and -7 are the most conserved . Binding experiments with a series of adenine analogues at position -11 revealed that the N1 nitrogen of adenine was a critical determinant for the preference of the adenine at this position, suggesting a mechanism for the nucleation of promoter DNA melting initiation in which RNA polymerase destabilizes duplex DNA by directly competing with the thymine of the A-T base pair for hydrogen bonding to the N1 position of the -11 nontemplate strand adenine.

Urol Res, 2000 Aug, 28(4), 269 - 73
Determination of mouse bladder inflammatory response to E . coli lipopolysaccharide; Jerde TJ et al.; Evaluation of the severity of histologic changes associated with cystitis is often subjective and inconsistent from one sample to the next . The objective of this study was to establish a consistent, reproducible method to quantify histologic changes in a mouse model of lipopolysaccharide (LPS)-induced cystitis . Either LPS (n = 8) or pyrogen-free saline (n = 8) was instilled intravesically into the bladders of female C57bk-6 J mice . Twenty-four hours later, mice in these groups as well as eight untreated controls were sacrificed and bladders were removed, fixed in formalin, and stained with hematoxylin and eosin (H&E) . A bladder inflammatory index (BII) was described by reviewing tissues for edema, leukocyte infiltration, and hemorrhage . Cross-sections were evaluated by a single pathologist in a blinded manner based on the objective BII described . The BII method for objectively analyzing bladder inflammation was effective and reproducible . Bladders instilled with LPS had significantly increased inflammation scores for edema, leukocyte infiltration, and hemorrhage compared with those instilled with saline or untreated controls (n = 8, P < 0.05) . These results demonstrate that LPS causes bladder inflammation when instilled intravesically and that inflammation of mouse bladders can be objectively quantified using the histological method described.

Artif Cells Blood Substit Immobil Biotechnol, 2000 Sep, 28(5), 397 - 408
Artificial cells microencapsulated genetically engineered E . coli DH5 cells for the lowering of plasma creatinine in-vitro and in-vivo; Prakash S et al.; High level of plasma creatinine occurs in renal insufficiency, uremia, and other diseases . At present lowering of this metabolite is done by using dialysis and other techniques . In this article, we report the use of artificial cells microencapsulated genetically engineered E . coli DH5 cells for lowering plasma creatinine in-vitro and in-vivo . Result shows that artificial cells were able to lower plasma creatinine in-vitro from 21.80 +/- 1.10 mg/dl to 21.80 +/- 1.10 mg/dl in 60 minutes and to 19.34 +/- 0.60 mg/dl in 3 hours . Result also shows that when given orally on a daily basis, artificial cells microencapsulated genetically engineered E . coli DH5 cells were also able to lower plasma creatinine in rats.

J Org Chem, 2000 Sep 22, 65(19), 5891 - 7
Probing the stereochemistry of E . coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (phenylalanine-sensitive)-catalyzed synthesis of KDO 8-P analogues; Sundaram AK et al.; The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-{3-(2)H}-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-{3-(2)H}-PEP (3S)-{3-(2)H}-3-deoxy-D-manno-octulosonate 8-phosphate, (3S)-{3-(2)H}-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-{3-(2)H}-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-{3-(2)H}-PEP gives the corresponding (3R)-monosaccharides . These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested . Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-{3-F}-PEP with E 4-P were completely characterized by (1)H and (19)F NMR analysis for the first time . Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.

Mol Cells, 2000 Aug 31, 10(4), 405 - 10
Characterization of recombinant Dictyostelium discoideum sepiapterin reductase expressed in E . coli; Kim YA et al.; A cDNA clone (SSC801) putatively encoding sepiapterin reductase (SR) was obtained from the expressed sequence tag clones of Dictyostelium discoideum . The cDNA sequence of 878 nucleotides constituted an ORF of 265 amino acid residues but was missing a few N-terminal residues . The deduced amino acid sequence showed 29.8% identity with mouse SR sequence and a molecular mass of 29,969 Da . The coding sequence was cloned in E . coli expression vector and overexpressed . The purified His-tag recombinant enzyme was confirmed to have the genuine activity of SR to produce tetrahydrobiopterin from 6-pyruvoyltetrahydropterin in a coupled assay with 6-pyruvoyltetrahydropterin synthase as well as dihydrobiopterin from sepiapterin . However, dictyopterin was not observed in our assay condition . The enzyme was also inhibited by N-acetylserotonin and to a lesser extent by melatonin . Km values for NADPH and sepiapterin were 51.8+/-2.7 microM and 40+/-2 microM, respectively . Vmax was determined as 0.14 micromol/min/mg of protein.

J Immunol Methods, 2000 Aug 28, 242(1-2), 101 - 14
Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E . coli; Schmiedl A et al.; New E . coli vectors based on the pOPE/pSTE vector system {Gene 128 (1993) 97} were constructed to express a single-chain Fv antibody fragment (scFv), a scFv-streptavidin fusion protein and two disulfide bond-stabilized Fv antibody fragments (dsFvs) utilizing different side chain positions for disulfide stabilization . All of these constructs encoded fusion proteins carrying five C-terminal histidine residues preceded by an unpaired cysteine . The influence of this cysteine, which was originally introduced to allow the chemical modification of the fusion proteins, was assessed by exchanging the two amino acids CysIle in front of the carboxy terminal His-tag to SerHis in all constructs . Yield and antigen-binding activity of the antibody constructs were compared after standard lab-scale periplasmic expression in Escherichia coli . The removal of the unpaired cysteine resulted in a significant increase in antigen-binding activity of the crude periplasmic extracts . Further, a three-five fold increase of yield and a significantly improved purity were observed after immobilized metal affinity chromatography (IMAC) with all four constructs.

Biochemistry, 2000 Sep 5, 39(35), 10761 - 9
Isolation, identification, and synthesis of gamma-butyrobetainyl-CoA and crotonobetainyl-CoA, compounds involved in carnitine metabolism of E . coli; Elssner T et al.; A still unknown low-molecular-mass cofactor essential for the activity of carnitine-metabolizing enzymes (e.g., L-carnitine dehydratase, crotonobetaine reductase) from E . coli has been purified to homogeneity from a cell-free extract of E . coli O44K74 . The purity of the cofactor was confirmed by HPLC analysis . Biosynthesis of the unknown compound was only observed when bacteria were cultivated anaerobically in the presence of L-carnitine or crotonobetaine . The determined properties, together with results obtained from UV-visible, (1)H NMR, and mass spectrometry, indicate that the compound in question is a new CoA derivative . The esterified compound was suggested to be gamma-butyrobetaine-a metabolite of carnitine metabolism of E . coli . Proof of structure was performed by chemical synthesis . Besides gamma-butyrobetainyl-CoA, a second new CoA derivative, crotonobetainyl-CoA, was also chemically synthesized . Both CoA derivatives were purified and their structures confirmed using NMR and mass spectrometry . Comparisons of structural data and of the chemical properties of gamma-butyrobetainyl-CoA, crotonobetainyl-CoA, and the isolated cofactor verified that the unknown compound is gamma-butyrobetainyl-CoA . The physical and chemical properties of gamma-butyrobetainyl-CoA and crotonobetainyl-CoA are similar to known CoA derivatives.

J Org Chem, 2000 Sep 8, 65(18), 5609 - 14
Synthesis and characterization of the native anticodon domain of E . coli TRNA(Lys): simultaneous incorporation of modified nucleosides mnm(5)s(2)U, t(6)A, and pseudouridine using phosphoramidite chemistry; Sundaram M et al.; The anticodon domain of E . coli tRNA(Lys) contains the hypermodified nucleosides mnm(5)s(2)U and t(6)A at positions 34 and 37, respectively, along with a more common psi at position 39 . The combination of these three nucleotides represents one of the most extensively modified RNA domains in nature . 2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm(5)s(2)U and t(6)A were each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis . The 17 nucleotide anticodon stem-loop of E . coli tRNA(Lys) was then assembled from these synthons using phosphoramidite coupling chemistry . Coupling efficiencies for the two hypermodified nucleosides and for pseudouridine phosphoramidite were all greater than 98% . A mild deprotection scheme was developed to accommodate the highly functionalized RNA . High coupling yields, mild deprotection, and efficient HPLC purification allowed us to obtain 1 . 8 mg of purified RNA from a 1 micromol scale RNA synthesis . Our efficient synthetic protocol will allow for biophysical investigation of this rather unique tRNA species wherein nucleoside modification has been shown to play a role in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal frameshifting . The human analogue, tRNA(Lys,3), is the specific tRNA primer for HIV-1 reverse transcriptase and has a similar modification pattern.

J Mol Biol, 2000 Sep 8, 302(1), 235 - 50
Complementation between dimeric mutants as a probe of dimer-dimer interactions in tetrameric dihydrofolate reductase encoded by R67 plasmid of E . coli; Dam J et al.; The effect of mutations on the interactions between dimers in R67 dihydrofolate reductase (R67 DHFR), a tetrameric enzyme conferring resistance to trimethoprim, was investigated by site-directed mutagenesis combined with phenotypic, enzymatic, and biochemical analysis.Some 14 mutants at two positions involved in a hydrogen bond between dimers were constructed . All were shown to be dimers . However, complementation between pairs of dimeric mutated proteins resulted in the restoration of the enzymatic activity and heterotetramer formation . A combinatorial approach was set up to create efficiently such heterotetramers and identify the complementing pairs of mutations . A dozen of such pairs were found . An accurate method was set up to measure the association of the complementing dimers in a "quasi-isologous" heterotetramer and used to study the effects of mutations and pH on the association . Thus, the pair of proteins bearing respectively the S59A and H62L mutations was shown to form heterotetramers with catalytic properties close to those of the wild-type protein . Its association was as strong as that of the wild-type protein at cytoplasmic pH (6 . 5), and was more stable at lower pH values.A double-mutant protein bearing simultaneously the S59A and H62L mutations was produced and analyzed . Its association was weakened by 1.2 kcal/mol as compared to the wild-type enzyme at pH 6.5 but was insensitive to pH . Comparing the energy of association between dimers in the wild-type protein, the heterotetramer and the double mutant allowed us to dissect the effects of the pH and of the molecular context on a subset of interactions between the R67 DHFR subunits .

Arch Virol, 2000, 145(7), 1305 - 20
Expression of stable hepatitis B viral polymerase associated with GRP94 in E . coli; Kim SS et al.; We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli . We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins . The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors . The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation . However, nondenaturing and partially denaturing activity gel analyses showed two active bands of approximately 260 kDa and approximately 130 kDa, respectively . Furthermore, in the presence of the encapsidation signal RNA template (HBV epsilon RNA), the approximately 260-kDa active band was gradually converted to approximately 130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV epsilon RNA . These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.

Peptides, 2000 Jun, 21(6), 767 - 72
Expression of functional recombinant scorpion beta-neurotoxin Css II in E . coli; Johnson TM et al.; The gene for a beta-neurotoxin {Centruroides suffusus suffusus toxin II (Css II)} from the scorpion C . suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b . This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG) . Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins . The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography . Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species . The recombinant neurotoxin inhibits the uptake of {(3)H}GABA {gamma-aminobutyric acid (GABA)} in neuronal cells as effectively as natural beta-toxins.

Genetica, 1999, 107(1-3), 181 - 7
Transposable elements as activators of cryptic genes in E . coli; Hall BG; The concept of transposable elements (TEs) as purely selfish elements is being challenged as we have begun to appreciate the extent to which TEs contribute to allelic diversity, genome building, etc . Despite these long-term evolutionary contributions, there are few examples of TEs that make a direct, positive contribution to adaptive fitness . In E . coli cryptic (silent) catabolic operons can be activated by small TEs called insertion sequences (IS elements) . Not only do IS elements make a direct contribution to fitness by activating cryptic operons, they do so in a regulated manner, transposing at a higher rate in starving cells than in growing cells . In at least one case, IS elements activate an operon during starvation only if the substrate for that operon is present in the environment . It appears that E . coli has managed to take advantage of IS elements for its own benefit.

Genes Dev, 2000 Aug 15, 14(16), 2097 - 105
Defective processing of methylated single-stranded DNA by E . coli AlkB mutants; Dinglay S et al.; Escherichia coli alkB mutants are very sensitive to DNA methylating agents . Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog . Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants . No such decrease occurred when using methylated lambda phage or M13 duplex DNA . These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA . Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA . The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by gamma irradiation . Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant . A recA mutation caused a small but additive defect . Thus, AlkB functions in a novel pathway independent of these activities . We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions . Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells.

Biotechniques, 2000 Aug, 29(2), 288 - 90, 292, 294 passim
Two-hybrid system for characterization of protein-protein interactions in E . coli; Hays LB et al.; The yeast two-hybrid system has been used to characterize many protein-protein interactions . A two-hybrid system for E . coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site . The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain . In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the lambda repressor . Reporters were designed to express cat and lacZ under the control of a low-affinity lambda operator . At low expression levels, lambda repressor hybrids weakly repress the reporter genes . A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone . If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the lambda repressor hybrid to the lambda operator, causing increased repression of the reporter genes . Reconstruction experiments with the fos and jun leucine zippers detected protein-protein interactions between either homodimeric or heterodimeric leucine zippers.

Mutat Res, 2000 Aug 30, 460(3-4), 257 - 75
Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E . coli Vsr protein, and human nucleotide excision repair factor XPA; Morikawa K et al.; Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones . The understanding of this repair mechanism is a central subject in cell biology . This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins . The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation . The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition . The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence . The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur . The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex . This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA . The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR . This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.

Mutat Res, 2000 Aug 30, 460(3-4), 201 - 10
Structural studies of human alkyladenine glycosylase and E . coli 3-methyladenine glycosylase; Hollis T et al.; Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA . The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis . Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases . Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond . In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.

J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 183 - 8
Expression and characterization of E . coli-produced soluble, functional human dihydroorotate dehydrogenase: a potential target for immunosuppression; Neidhardt EA et al.; Human dihydroorotate dehydrogenase (huDHODH) is essential for de novo biosynthesis of pyrimidines and the target of two immunosuppressive drugs, brequinar and the leflunomide metabolite A77-1726 (Chen et al., 1992; Davis et al., 1996) . Using a T7 RNA polymerase expression system, we produced huDHODH as a fusion protein containing an amino-terminal decahistidine tag . Escherichia coli growth and expression conditions were optimized to enhance huDHODH solubility and to permit purification of the enzyme in the absence of detergent . Soluble huDHODH, purified by a simple two-step procedure, was catalytically active, monomeric, and contained a flavin mononucleotide (FMN) cofactor in a 1:1 FMN/protein molar ratio . Kinetic analysis showed that huDHODH uses a two site ping-pong mechanism, where DHO is oxidized at one site and the second substrate, ubiquinone, is reduced at the other . This result is consistent with the mechanism proposed for bovine liver DHODH (Hines and Johnston, 1989).

Int J Artif Organs, 2000 Jul, 23(7), 429 - 35
In vitro and in vivo uric acid lowering by artificial cells containing microencapsulated genetically engineered E . coli DH5 cells; Prakash S et al.; Increase in systemic uric acid occurs in renal insufficiency, gout, chemotherapy, and other diseases . Dialysis can lower this metabolite but is expensive . The use of drugs can, sometime, result in side effects . Therefore, a suitable affordable method for this is required . In this article, for the first time, we report the use of artificial cells containing micro encapsulated genetically engineered E . Coli DH5 cells for lowering uric acid in vitro and in vivo . Results show that this novel approach has the ability to significantly lower uric acid from 84.80 +/- 3.40 mg/dl to 9.32 +/- 0.05 mg/dl in vitro and from the plasma of the experimental animals from the control levels of 71.00 +/- 27.49 mg/dl to 20.33 +/- 17.92 mg/dl in vivo . Continued daily oral administration maintained the plasma uric acid concentration of experimental uremic rats to the normal plasma uric acid level range during the entire test period.

J Mol Microbiol Biotechnol, 2000 Jul, 2(3), 283 - 9
OmpT expression and activity increase in response to recombinant chloramphenicol acetyltransferase overexpression and heat shock in E . coli; Gill RT et al.; The activity of a 35 kDa protease increased in response to induced expression of chloramphenicol acetyltransferase (CAT) in E . coli . This protease was partially purified, extensively characterized, and identified via the use of zymogram gels as the outer membrane protease, OmpT . In experiments targeting the overlap of well-characterized stress responses, OmpT activity was found to increase in response to heat shock but was only minimally affected by amino acid limitation . The largest increase in activity was found after induction of CAT . OmpT expression levels also increased in response to induction of recombinant CAT overexpression and heat shock . This is the first report of increased activity and expression of an outer membrane protease during cytoplasmic overexpression of a recombinant protein.

J Mol Microbiol Biotechnol, 2000 Jul, 2(3), 261 - 3
ATPase and GTPase activities copurifying with GTP-binding proteins in E . coli; Sayed A et al.; Intrinsic GTPase activity of GTP-binding proteins plays the vital role in regulating the downstream activation pathway . We examined the GTP and ATP hydrolyzing (NTPase) abilities of various bacterial and human GTP-binding proteins under different metabolic conditions . Two metabolic components, acetate and 3-phosphoglyceric acid (3-PG), have shown significant stimulatory action on NTPase activity of G-protein preparations . Acetyl phosphate and 2,3-bisphosphoglyceric acid (2,3-BPG) blocked these stimulations . From gel filtration analyses, we have determined two fractions containing metabolite-inducible NTPase activities which are independent of GTP-binding protein enzymatic actions . Therefore, one should be cautious when NTPase activity is examined in a buffer containing acetate often used for NTPase assay.

Nat Struct Biol, 2000 Aug, 7(8), 648 - 52
Structure of the DNA binding domain of E . coli SSB bound to ssDNA; Raghunathan S et al.; The structure of the homotetrameric DNA binding domain of the single stranded DNA binding protein from Escherichia coli (Eco SSB) bound to two 35-mer single stranded DNAs was determined to a resolution of 2.8 A . This structure describes the vast network of interactions that results in the extensive wrapping of single stranded DNA around the SSB tetramer and suggests a structural basis for its various binding modes.

J Mol Biol, 2000 Aug 11, 301(2), 257 - 64
Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E . coli ribonuclease E; Kaberdin VR et al.; Endonucleolytic cutting by the essential Escherichia coli ribonuclease RNaseE has a central role in both the processing and decay of RNA . Previously, it has been shown that an oligoribonucleotide corresponding in sequence to the single-stranded region at the 5' end of RNAI, the antisense regulator of ColE1-type plasmid replication, is efficiently cut by RNaseE . Combined with the knowledge that alteration of the structure of stem-loops within complex RNaseE substrates can either increase or decrease the rate of cleavage, this result has led to the notion that stem-loops do not serve as essential recognition motifs for RNaseE, but can affect the rate of cleavage indirectly by, for example, determining the single-strandedness of the site or its accessibility . We report here, however, that not all oligoribonucleotides corresponding to RNaseE-cleaved segments of complex substrates are sufficient to direct efficient RNaseE cleavage . We provide evidence using 9 S RNA, a precursor of 5 S rRNA, that binding of structured regions by the arginine-rich RNA- binding domain (ARRBD) of RNaseE can be required for efficient cleavage . Binding by the ARRBD appears to counteract the inhibitory effects of sub-optimal cleavage site sequence and overall substrate conformation . Furthermore, combined with the results from recent analyses of E . coli mutants in which the ARRBD of RNase E is deleted, our findings suggest that substrate binding by RNaseE is essential for the normal rapid decay of E . coli mRNA . The simplest interpretation of our results is that the ARRBD recruits RNaseE to structured RNAs, thereby increasing the localised concentration of the N-terminal catalytic domain, which in turn leads to an increase in the rate of cleavage .

Exp Mol Med, 2000 Jun 30, 32(2), 93 - 9
Properties of GST-CALM expressed in E . coli; Kim JA et al.; Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells . CCVs are composed of clathrin and assembly proteins . The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180 . In this study, we characterized the properties of the CALM expressed in E . coli . The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE . The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage . However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages . The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro . The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.

Zhonghua Yi Xue Za Zhi, 1998 Apr, 78(4), 308 - 10
{Human bone morphogenetic protein-2: recombinant expression in E . coli}; Ma Q et al.; OBJECTIVE: To explore the way of producing human bone morphogenetic protein-2(hBMP-2) for bone healing by using the gene engeneering techniques . METHODS: hBMP-2 cDNA fragment, which consists of 3' end partial propeptide and mature peptide sequence, was inserted into the multiple cloning site of expression vector pkpL-3a via ligation . The recombinant plasmid pkpL-3a/hBMP-2 was transformed into E . coli pOp2136 . By the method of restriction map, the positive expression clone was selected . SDS-PAGE analysis showed a new foreign protein band near 27,000 after induction . The yield of induced hBMP-2 accouned for 10% of the total bacterial proteins . RESULTS: The partial purified recombinant hBMP-2 was implanted into Wistar rat thigh . After 4 weeks, histological analysis showed that it induced the proliferation of mesenchymal type cells and formation of new cartilage and bone in the implantation area . CONCLUSION: The hBMP-2 produced by gene engeneering techniques has the biologic capacity of ectopic bone formation.

J Biochem (Tokyo), 2000 Aug, 128(2), 309 - 13
Studies on ATPase(GTPase) intrinsic to E . coli ribosomes; Ogata K et al.; (1) Escherichia coli 70S ribosomes showed intrinsic ATPase and GTPase activities, although they were much lower than those of rat liver ribosomes . The latter activity was higher than the former one . (2) The ATPase activity was inhibited by GTP and GMP-P(NH)P, and the GTPase activity was inhibited by ATP and AMP-P(NH)P, indicating a close relationship between the two enzymes . (3) Elongation components alone or in combination enhanced the ATPase activity, indicating the possible correlation of ribosomal ATPase with elongational components . (4) Vanadate at the concentrations that did not inhibit the GTPase activities of EF-Tu and EF-G, depressed the poly(U)-dependent polyphe synthesis, suggesting that ribosomal ATPase (GTPase) participates in peptide elongation by inducing positive conformational changes of ribosomes required for the attachment of elongational components.

Harefuah, 1998 Dec 1, 135(11), 503 - 4, 567
{Normotensive hydrocephalus complicating recurrent E . coli meningitis}; Zamir D et al.; E . coli meningitis is a disease that occurs in predisposed patients, either as a result of trauma or in neonates after neurosurgery . Recurrent E . coli meningitis in an adult without any apparent predisposition is uncommon, and hydrocephalus complicating bacterial meningitis is even more rare . We report a unique case of a 67-year-old alcoholic man who had had 2 consecutive episodes of E . coli meningitis within 2 months . In both episodes there was a favorable response to ceftriaxone . However, normotensive hydrocephalus appeared a few weeks later, with mental and physical deterioration.

Harefuah, 1998 May 15, 134(10), 767 - 9, 831
{Acute gastroenteritis caused by enterohemorrhagic E . coli O157:H7}; Weinberger T et al.; We report a 48-year-old man admitted for watery diarrhea, high fever, chills and abdominal cramps . Enterohemorrhagic E . coli O157:H7 was isolated . This new, dangerous pathogen causes dysentery and complications such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura . These complications can cause renal failure, neurological deficit and death . Recognition of E . coli O157:H7 infection is important since it causes a rare and dangerous condition . To the best of our knowledge this is the first case reported in Israel.

Biotechniques, 2000 Jul, 29(1), 146 - 54
The admid system: generation of recombinant adenoviruses by Tn7-mediated transposition in E . coli; Richards CA et al.; A new system has been developed for generating recombinant adenoviruses by Tn7-mediated transposition in E . coli . Low copy number E . coli plasmids containing a full-length adenoviral genome with lacZattTn7 replacing E1 have been constructed . The adenovirus plasmid or admid, as well as high copy number progenitors, were stably maintained in E . coli strain DH10B . Several transfer vectors containing a mammalian expression cassette flanked by Tn7R and Tn7L were used as donors to transpose the mini-Tn7 into the E1 region of the adenoviral genome . Transposed recombinant admids are readily identified by their beta-galactosidase phenotype . Transfection of admid DNA into producer cells resulted in the efficient production of infectious adenovirus . This easy-to-use, efficient system generates pure, clonal stocks of recombinant adenovirus without successive rounds of plaque purification.

Anal Chem, 2000 Jul 1, 72(13), 2981 - 6
Intensities of E . coli nucleic acid Raman spectra excited selectively from whole cells with 251-nm light; Wu Q et al.; Escherichia coli bacteria in the logarithmic growth phase have been investigated by UV resonance Raman spectroscopy . Bacterial whole-cell Raman spectra excited at 251 nm reflect nearly exclusively the nucleic acid composition even though a very large fraction of the bacterial mass is composed of protein . It has been demonstrated that if bacteria are grown under controlled (logarithmic growth) conditions, which give rise to organisms of known average biochemical composition, the intensities of E . coli Raman spectra can be explained quantitatively from the knowledge of component nucleic acid base resonance Raman cross sections.

Arch Biochem Biophys, 2000 Aug 1, 380(1), 11 - 9
Positively charged residues within the iron-sulfur cluster loop of E . coli MutY participate in damage recognition and removal; Chepanoske CL et al.; Escherichia coli MutY is an adenine glycosylase involved in base excision repair that recognizes OG:A (where OG = 7, 8-dihydro-8-oxo-2'-deoxyguanosine) and G:A mismatches in DNA . MutY contains a solvent-exposed polypeptide loop between two of the cysteine ligands to the {4Fe-4S}(2+) cluster, referred to as the iron-sulfur cluster loop (FCL) motif . The FCL is located adjacent to the proposed active site pocket and has been suggested to be part of the DNA binding surface of MutY (Y . Guan et al., 1998, Nat . Struct . Biol . 5, 1058-1064) . In order to investigate the role of specific residues within the FCL motif, we have determined the effects of replacing arginine 194, lysine 196, and lysine 198 with alanine on the enzymatic properties of MutY . The properties of the R194A, K196A, and K198A enzymes were also compared to the properties of mutated enzymes in which lysine residues near the active site pocket were replaced with alanine or glycine . Substrate recognition was evaluated using a duplex containing a 2'-deoxyadenosine analog in a base pair opposite G or OG . These results indicate that removal of positively charged amino acids within the FCL and the active site compromise the ability of the enzyme to bind to the substrate analog . However, only the K198A enzyme exhibited a significant reduction (15-fold) of the rate of adenine removal from a G:A base pair-containing duplex . This is the first direct evidence that Lys 198 within the FCL motif of MutY has a role in specific damage recognition and removal . Furthermore, these results suggest that the FCL motif is intimately involved in the base removal process .

Genes Dev, 2000 Jul 15, 14(14), 1734 - 40
Bcl-2 antiapoptotic protein mediates verotoxin II-induced cell death: possible association between bcl-2 and tissue failure by E . coli O157:H7; Suzuki A et al.; Verotoxin II (VTII: or Shiga-like toxin 2) is a key factor for Escherichia coli O157:H7-induced multiple tissue failure and contains a pentameric sequence (NWGRI) similar to the Bcl-2 homolog domain, BH1 . In the current study, we demonstrate that VTII, but not VTI, interacts with Bcl-2 through each BH1 domain pentameric sequence (NWGRI) and that the VTII/Bcl-2 complex is necessary for cell-death induction in target cells . VTII translocates to mitochondria and induces cell death only when target cells are expressing Bcl-2 . In addition, interruption of VTII-Bcl-2 complex formation by a pentameric BH1 synthetic peptide suppresses VTII-induced cell death . In the present article, we propose that Bcl-2 mediates VTII-induced target cell death by the interaction with each pentameric sequence of BH1 domain.

Thromb Haemost, 2000 Jun, 83(6), 840 - 3
Prothrombotic risk factors in children with acute lymphoblastic leukemia treated with delayed E . coli asparaginase (COALL-92 and 97 protocols); Mauz-Korholz C et al.; Hereditary prothrombotic risk factors have been shown to increase the risk of venous thrombosis in children treated with the combination of E . coli asparaginase and steroids . In the present study the role of prothrombotic risk factors in children with ALL treated according to the COALL study protocol was investigated in 108 consecutively recruited childhood patients . The prevalence rates of prothrombotic risk factors {factor V G1691A mutation, the prothrombin G20210A variant, the TT677 methylenetetrahydrofolate reductase genotype, deficiencies of protein C, protein S, antithrombin, elevated lipoprotein (a)} in this cohort were within the range reported for healthy Caucasians, and comparable to previously reported data for other leukemic patients . Venous thromboembolism occurred in 3 of the 108 children (induction n = 1; reinduction n = 2: 2.8%), and none of these children carried a prothrombotic risk factor . The results of the present study, suggest that the role of hereditary and acquired disturbances of coagulation in the development of thromboses might depend on the treatment regimen.

Am J Physiol Lung Cell Mol Physiol, 2000 Jul, 279(1), L100 - 9
E . coli hemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid; Rose F et al.; Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury . In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge . In parallel, large quantities of leukotriene (LT) B(4) and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply . Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB(4) and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB(5) and 5-, 8-, 9-, and 12-hydroxyeicosapentaenoic acid (HEPE) . At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB(5) and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB(4) and 5-, 8-, 9-, and 12-HETE by >80-fold . In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE . We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis . In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.

Cell, 2000 Jun 9, 101(6), 613 - 23
6S RNA regulates E . coli RNA polymerase activity; Wassarman KM et al.; The E . coli 6S RNA was discovered more than three decades ago, yet its function has remained elusive . Here, we demonstrate that 6S RNA associates with RNA polymerase in a highly specific and efficient manner . UV crosslinking experiments revealed that 6S RNA directly contacts the sigma70 and beta/beta' subunits of RNA polymerase . 6S RNA accumulates as cells reach the stationary phase of growth and mediates growth phase-specific changes in RNA polymerase . Stable association between sigma70 and core RNA polymerase in extracts is only observed in the presence of 6S RNA . We show 6S RNA represses expression from a sigma70-dependent promoter during stationary phase . Our results suggest that the interaction of 6S RNA with RNA polymerase modulates sigma70-holoenzyme activity.

Biochemistry, 2000 Jul 11, 39(27), 8058 - 66
A cis-proline to alanine mutant of E . coli aspartate transcarbamoylase: kinetic studies and three-dimensional crystal structures; Jin L et al.; The only cis-proline residue in Escherichia coli aspartate transcarbamoylase has been replaced by alanine using site-specific mutagenesis . The Pro268-->Ala enzyme exhibits a 40-fold reduction in enzyme activity and decreased substrate affinity toward carbamoyl phosphate and aspartate compared to the corresponding values for the wild-type enzyme . The concentration of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) required to activate the mutant enzyme to the same extent as the wild-type enzyme is significantly increased . The heterotropic effects of ATP and CTP upon the Pro268-->Ala enzyme are also altered . Crystal structures of the Pro268-->Ala enzyme in both T- and R-states show that the cis-peptidyl linkage between Leu267 and Ala268 is maintained . However, the tertiary structure of both the catalytic and regulatory chains has been altered by the amino acid substitution, and the mobility of the active-site residues is increased for the R-state structure of Pro268-->Ala enzyme as comparison with the wild-type R-state structure . These structural changes are responsible for the loss of enzyme activity . Thus, Pro268 is required for the proper positioning of catalytically critical residues in the active site and is important for the formation of the high-activity high-affinity R-state of E . coli aspartate transcarbamoylase.

Biochemistry (Mosc), 2000 Jun, 65(6), 640 - 50
Probing contacts of phosphate groups of oligonucleotides from the non-template strand of lac UV5 promoter with E . coli RNA polymerase using regioselective cross-linking; Rudakova EA et al.; Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 promoter with lysines or histidines of E . coli RNA polymerase in the open complex model were studied . A number of synthetic oligonucleotides from the -10-area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase . 5'-N-Hydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosphate activated derivatives . They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into proximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein . The analysis of the products of cross-linking allowed the protein subunit and the amino acid residue taking part in the formation of the covalent bond for each oligonucleotide to be identified . It was found that all oligonucleotides from the non-template strand of promoter in the complex with the holo-enzyme are bound with the sigma70-subunit . When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with His180 or His242 of the sigma70-subunit, the reactive amino acid residue being located between the first and second conservative regions . Phosphate at position -15 is located near lysines from two different areas--between Met413 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3.1) . Phosphate at position -18 makes preferential contacts with a lysine situated between Met470 and Met507 (region 3.1) . Based on the analysis of contacts of phosphate groups and the structure of the isolated sigma70-subunit established previously, a scheme of the mutual arrangement of the oligonucleotide and the sigma70-subunit possessed by the holo-enzyme has been proposed.

Zhonghua Gan Zang Bing Za Zhi, 2000 Jun, 8(3), 171 - 3
Expression of human single-chain variable fragment antibody against non-structural protein 3 of hepatitis C virus antigen in e.coli; Zhong Y et al.; OBJECTIVE: To express human single-chain variable fragment (ScFv) antibody against non-structural protein 3 (NS(3)) of hepatitis C virus in E.coli . METHODS: The recombinant phages were panned by NS(3) antigen which was coated in a microtiter plate . After five rounds of biopanning, 66 clones were identified specific to NS(3) antigen . E.coli host XL(1)-Blue was transformed and induced by IPTG . The specificity of ScFv was evaluated by ELISA and dot blot hybridization . RESULTS: ScFv-NS(3) DNA digestion and sequencing data showed that the ScFv gene was composed of 750bp . ELISA and dot blot hybridization demonstrated that the soluble human single-chain Fv antibody to hepatitis C virus NS(3) antigen could combine different origins of NS(3) antigen . CONCLUSION: NS(3)-ScFv antibody expressed by E.coli host XL(1)-Blue has the activity and specificity to combine different origins of HCV NS(3) antigen.

Mol Gen Mikrobiol Virusol, 2000, (2), 33 - 6
{Integration of plasmids into E . coli K12 chromosomes, caused by a Bordetella transposon}; Sivov IG et al.; Transposition of Bordetella pertussis transposon in E . coli chromosome has been studied on a model of exclusion of donor multicopy pKK3 plasmid with coumermicin . TnBP3 induced the formation of co-integrates between the plasmid and chromosome . The structure of co-integrate was determined . Facts of exclusion of integrated structure and transposon transposition within integrated plasmid into new sites on a recipient chromosome were detected . Relationship between these processes and activity of bacterial cell recombination system has been determined.

EMBO J, 1983, 2(8), 1271 - 4
72 residues of gal repressor fused to beta-galactosidase repress the gal operon of E . coli; von Wilcken-Bergmann B et al.; An active gene has been constructed which produces a chimera consisting of the N-terminal domain of the gal repressor and all but the first five residues of beta-galactosidase . Seventy two residues of gal repressor fused to beta-galactosidase as tetrameric core are sufficient to repress the gal operon in vivo and to bind to the gal operator in vitro.

Eur J Epidemiol, 2000 Mar, 16(3), 303 - 4
Incidence of E . coli O157:H7 and other enteropathogens in a Spanish hospital; Lopez AG et al.; From January to December 1997, stools submitted for routine culture were also screened for E . coli O157:H7 to investigate its incidence in our area . Bloody and non-bloody stools were studied . E . coli O157:H7 was not recovered from any of the samples tested . We believe, therefore, that we are in a low rate of infection area, and accordingly do not recommend routine screening of this pathogen.

Nature, 2000 Jun 8, 405(6787), 694 - 7
Alpha-haemolysin of uropathogenic E . coli induces Ca2+ oscillations in renal epithelial cells; Uhlen P et al.; Pyelonephritis is one of the most common febrile diseases in children . If not treated appropriately, it causes irreversible renal damage and accounts for a large proportion of end stage renal failures . Renal scarring can occur in the absence of inflammatory cells, indicating that bacteria may have a direct signalling effect on renal cells . Intracellular calcium ({Ca2+}i) oscillations can protect cells from the cytotoxic effects of prolonged increases in intracellular calcium . However, no pathophysiologically relevant protein that induces such oscillations has been identified . Here we show that infection by uropathogenic Escherichia coli induces a constant, low-frequency oscillatory {Ca2+}i response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin alpha-haemolysin . The response depends on calcium influx through L-type calcium channels as well as from internal stores gated by inositol triphosphate . Internal calcium oscillations induced by alpha-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8 . Our findings indicate a novel role for alpha-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.

Bull Soc Pathol Exot, 2000 Apr, 93(2), 95 - 6
{Isolation of enteric pathogeic agents in Côte d'Ivoire: Escherichia coli O157:H7 and enteroaggregative E . coli}; Dadie A et al.; New pathogens including Escherichia coli O157:H7 have emerged and spread world-wide as the most important cause of foodborne infections . We established a prospective study in Abidjan from 1996 to 1999 to determine the prevalence of Shiga-toxin producing E . coli (STEC) in our environment . Two O157 strains were found . One (EA47) O157:H7 was isolated from chicken and the other (EH144) O157:HNM from human diarrhoeal stool specimens . Both O157 strains carried stx2, eae, and UidA genes, but not e-hly one . Four other pathogenic E . coli were isolated, including three enteroaggregative E . coli (EAggEC) and one isolate which expresses a cytolethal distending toxin gene (cdtB) . This is the first report of Shiga-toxin producing E . coli (STEC) in Cote d'Ivoire . Given its low prevalence (0.8%), E . Coli does not appear to be a public health problem in Cote d'Ivoire.

J Mol Biol, 2000 Jun 2, 299(2), 379 - 89
Mutations at position A960 of E . coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA; Sergiev PV et al.; The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments . Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA . Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon . Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself . These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation .

J Mol Biol, 2000 May 26, 299(1), 1 - 15
Interaction of translation initiation factor IF1 with the E . coli ribosomal A site; Dahlquist KD et al.; Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli . However, the precise function of IF1 remains unknown . Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA . IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes . The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS) . The N1-N2 positions of G530 are also protected from reactivity with kethoxal . Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics . IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal . To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers . The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding . Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations . IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.

J Virol Methods, 2000 Jun, 87(1-2), 109 - 22
Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E . coli-expressed nucleocapsid protein as antigen; Dea S et al.; The 15 kDa nucleocapsid (N) protein is the most abundant protein of the porcine reproductive and respiratory syndrome virus (PRRSV), and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease . In this study, complementary DNA corresponding to the entire N gene of the IAF-Klop strain of PRRSV was cloned into the pGEX-4T-1 vector, and the N protein was expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein . The resulting GST-N recombinant fusion protein was purified by affinity chromatography and used as antigen for serological testing by indirect enzyme-linked immunosorbent assay (ELISA) . Two anti-N specific monoclonal antibodies (MAbs) (IAF-K8 and IAF-2B4), obtained following fusion experiments with spleen cells of BAlb/c mice that were immunized with the purified virus, were used in a competitive assay to increase the specificity of the ELISA . Both MAbs were found to be directed against highly conserved conformational epitopes of North American isolates of PRRSV . Optimal concentration of GST-N protein was determined by checkerboard titration, using hyperimmune pig antiserum to the homologous PRRSV strain, and corresponded to a range of 0.1-0.5 microg protein per well . When tested on 95 sera from pigs that were experimentally infected with the IAF-Klop strain, the competitive ELISA (K8-ELISA) was capable of detecting anti-PRRSV antibodies in 86.7% (65/75) and 92.6% (63/68) of pig sera known to be seropositive by indirect immunofluorescence (antibody titers >16) and a currently used commercial ELISA (HerdCheck(R); Idexx), with specificity values of 100 and 96.2%, respectively . When tested on clinical samples (542 sera) from 28 positive and 28 negative pig herds, the K8-ELISA performed in a similar way to HerdCheck(R) and immunofluorescence (IF) tests as shown by kappa values of 0.762 and 0.803 . The sensitivity and specificity of K8-ELISA were 100% on a herd basis, whereas sensitivity values of 80 and 82% with a specificity of 98.7% were determined on an individual basis in comparison with HerdCheck(R) and IF tests.

Acta Pol Pharm, 2000 Jan-Feb, 57(1), 49 - 52
Effects of iron and copper ions on the inducibility of hydrogen peroxide in E . coli; Marczewska J et al.; Inducing capacities of hydrogen peroxide in SOS chromotest were investigated in the absence and in the presence of iron and copper ions . Strain PQ37 of Escherichia coli and two strains with altered protection against oxidative damage (PQ300 and OG400) were employed . The latter two strains were more sensitive to the hydrogen peroxide inducing activity than strain PQ37 . At the concentration of 150 nmol/ml and 300 nmol/ml, iron ions induced SOS response . At the concentration range from 30 nmol/ml to 300 nmol/ml iron enhanced the effects produced by H2O2 . Results with copper were negative.

Int J Cancer, 2000 Jun 15, 86(6), 848 - 54
Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E . coli nitroreductase; Weedon SJ et al.; The enzyme nitroreductase from E . coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA . Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954 . We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells . Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx . 2, 000-fold sensitisation) with 25-50 p.f.u . per cell . Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines . Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation . Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours . In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001) .

J Biochem (Tokyo), 2000 Jun, 127(6), 977 - 83
Production of functional human selenocysteine-containing KDRF/thioredoxin reductase in E . coli; Koishi R et al.; In a previous study, we reported the isolation of a cDNA encoding KDRF (KM-102-derived reductase like factor) from the human bone marrow-derived stromal cell line KM-102 . Analysis of the sequence of this cDNA revealed it to be the previously reported human thioredoxin reductase cDNA . Human thioredoxin reductase, which was recently isolated from human lung adenocarcinoma NCI-H441 cells as a selenocysteine-containing selenoprotein, and its substrate thioredoxin are thought to be essential for protecting cells from the damage caused by reactive oxygen species . To obtain the selenocysteine-containing recombinant KDRF/thioredoxin reductase, we introduced a secondary structure, which is identical to the selenocysteine insertion signal of Escherichia coli formate dehydrogenase H mRNA, downstream of the TGA in the KDRF/thioredoxin reductase cDNA and expressed it in E . coli . As a result, a significant amount of selenocysteine was incorporated into the C-terminus of the KDRF/thioredoxin reductase protein . The selenocysteine-containing KDRF/thioredoxin reductase showed reducing activities toward human and E . coli thioredoxin, whereas non-selenocysteine-containing KDRF/thioredoxin reductase showed no enzyme activity . Our results suggest that this strategy will be applicable to the production of other mammalian selenocysteine-containing selenoproteins in E . coli.

J Inorg Biochem, 2000 Apr, 79(1-4), 59 - 65
New mechanistic insights into the reactivity of the R2 protein of E . coli ribonucleotide reductase (RNR); Twitchett MB et al.; Further to a linear free-energy correlation of cross-reaction rate constants k12 for the reaction of eight organic radicals (OR), e.g . MV*+, from methyl viologen, with cytochrome c(III), we consider here similar studies for the reduction of the R2 protein of Escherichia coli ribonucleotide reductase, which has FeIII2 and Tyr* redox components . The same two techniques of pulse radiolysis and stopped-flow were used . Cross-reaction rate constants (22 degrees C) at pH 7.0, I=0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E0(1) from -0.446 to +0.194 V . Samples of active-R2 have an FeIII2 met-R2 component, which in the present studies was close to 40% . Concurrent reactions have to be taken into account for the five most reactive ORs, corresponding to reduction of the FeIII2 of met-R2 and then of active-R2 . Separate experiments on met-R2 reproduced the first of these rate constants, which on average is approximately 66% larger than the second rate constant . A single Marcus free-energy plot of log k12-0.5 log10f versus -E0(1)/0.059 describes all the data and the slope of 0.54 is in satisfactory agreement with the theoretical value of 0.50 . Such behaviour is unexpected since the Tyr* is a much stronger oxidant (E0 approximately 1.0 V versus NHE) as compared to FeIII2 (E0 close to zero) . X-ray structures of the met- and red-R2 states have indicated that electroneutrality of the approximately 10 A buried active site is maintained . Proton transfer is therefore proposed as a rapid sequel to electron transfer . Other reactions considered are the much slower conventional time-range reductions of active-R2 with hydrazine and dithionite . For these reactions one and/or two-equivalent changes are possible . With both reductants, met-R2 reacts about four-fold faster than active-R2, and as with the ORs the less strongly oxidising FeIII2 component is reduced before the Tyr*.

Drug Dev Ind Pharm, 2000 Jun, 26(6), 661 - 9
Calibration and LOD/LOQ estimation of a chemiluminescent hybridization assay for residual DNA in recombinant protein drugs expressed in E . coli using a four-parameter logistic model; Lee KR et al.; Calibration is the process of fitting a model based on reference data points (x, y), then using the model to estimate an unknown x based on a new measured response, y . In DNA assay, x is the concentration, and y is the measured signal volume . A four-parameter logistic model was used frequently for calibration of immunoassay when the response is optical density for enzyme-linked immunosorbent assay (ELISA) or adjusted radioactivity count for radioimmunoassay (RIA) . Here, it is shown that the same model or a linearized version of the curve are equally useful for the calibration of a chemiluminescent hybridization assay for residual DNA in recombinant protein drugs and calculation of performance measures of the assay.

Gut, 2000 Jun, 46(6), 806 - 12
Do eicosanoids cause colonic dysfunction in experimental E coli O157:H7 (EHEC) infection?
Bell CJ, Elliott EJ, Wallace JL, Redmond DM, Payne J, Li Z, O'Loughlin EV.
BACKGROUND: The pathophysiology of enterohaemorrhagic Escherichia coli (EHEC) infection remains unclear . Eicosanoids have been implicated as pathophysiological mediators in other colitides . AIMS: To determine if prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) contribute to mucosal inflammation and dysfunction in EHEC colitis . METHODS: Ten day old rabbits were infected with EHEC . For five days after infection, mucosal synthesis of PGE(2) and LTB(4) was measured in distal colonic tissue from control and infected animals and (51)Cr-EDTA permeability was assessed in vivo . Myeloperoxidase activity was measured and histological inflammation and damage were assessed at five days in control and infected animals and after treatment of infected animals with the LTB(4) synthesis inhibitor MK-886 . In separate experiments, ion transport was measured in Ussing chambers, before and after in vitro addition of the cyclooxygenase inhibitor indomethacin . RESULTS: LTB(4) synthesis was increased from day 2 after infection onwards and PGE(2) synthesis was increased on day 3 . Mucosal permeability did not increase until day 5 after infection . MK-886 inhibited colonic LTB(4) production but did not reduce diarrhoea, inflammation, or mucosal damage . Electrolyte transport was not significantly altered on day 3 after infection . However, both Cl secretion and reduced Na absorption found on day 5 were partially reversed by indomethacin . CONCLUSIONS: Tissue synthesis of PGE(2) and LTB(4) did not correlate temporally with EHEC induced inflammation or changes in mucosal permeability and ion transport . Cyclooxygenase inhibition partially reversed ion transport abnormalities but lipoxygenase inhibition did not affect mucosal inflammation or histological damage . We conclude that the contribution of eicosanoids to mucosal injury and dysfunction is more complex than previously suggested.

J Tongji Med Univ, 1998, 18(3), 129 - 33
Effect of fragment Asp1961-Glu1978 in fibronectin on the expression of triple-domain polypeptide in E . coli; Li D et al.; Two plasmids were constructed and used to express two triple-domain recombinant polypeptide of human fibronectin (FN) . The cDNAs in plasmids code for two polypeptides, CH62 (Pro1239-Ser1515 of FN linked with Ala1690-Val2049 through Met) and CH63 (CH62 without Ile1850-Glu1978) . The expression level of CH62 in E . coli was very low, but that of CH63 was very high . The results suggests that Asp1961-Glu1978 in FN is a key sequence influencing the expression of triple-domain polypeptide in E . Coli . After being dissolved and renatured, CH63 can be purified by heparin-agarose affinity chromatography . Both of the cell-binding domains in the recombinant polypeptide were functional . The production of CH63 provides a fundamental basis for further study of recombinant products with better anti-metastasis function.

Nature, 2000 Apr 27, 404(6781), 1014 - 8
Roles of E . coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis; Tang M et al.; The expression of the Escherichia coli DNA polymerases pol V (UmuD'2C complex) and pol IV (DinB) increases in response to DNA damage . The induction of pol V is accompanied by a substantial increase in mutations targeted at DNA template lesions in a process called SOS-induced error-prone repair . Here we show that the common DNA template lesions, TT (6-4) photoproducts, TT cis-syn photodimers and abasic sites, are efficiently bypassed within 30 seconds by pol V in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol III's processivity beta,gamma-complex . There is no detectable bypass by either pol IV or pol III on this time scale . A mutagenic 'signature' for pol V is its incorporation of guanine opposite the 3'-thymine of a TT (6-4) photoproduct, in agreement with mutational spectra . In contrast, pol III and pol IV incorporate adenine almost exclusively . When copying undamaged DNA, pol V exhibits low fidelity with error rates of around 10(-3) to 10(-4), with pol IV being 5- to 10-fold more accurate . The effects of RecA protein on pol V, and beta,gamma-complex on pol IV, cause a 15,000- and 3,000-fold increase in DNA synthesis efficiency, respectively . However, both polymerases exhibit low processivity, adding 6 to 8 nucleotides before dissociating . Lesion bypass by pol V does not require beta,gamma-complex in the presence of non-hydrolysable ATPgammaS, indicating that an intact RecA filament may be required for translesion synthesis.

J Clin Microbiol, 2000 May, 38(5), 1866 - 8
Performance of the ImmunoCard STAT! E . coli O157:H7 test for detection of Escherichia coli O157:H7 in stools; Mackenzie A et al.; ImmunoCard STAT! E . coli O157:H7 (Meridian Diagnostics, Inc., Cincinnati, Ohio) is a novel rapid (10-min) test for the presence of Escherichia coli O157:H7 in stools . The test may be performed either directly on stool specimens or on an overnight broth culture of stool . In a multicenter prospective study, 14 of 14 specimens positive by culture for E . coli O157:H7 were positive by the ImmunoCard STAT! O157:H7 test, and there were no false positives from 263 culture-negative specimens . In a retrospective study, the test was positive in 339 (81%) of 417 stored culture-positive specimens and the specificity was 95% (98 of 103 specimens) . No false positives were associated with alternate stool pathogens . The ImmunoCard STAT! O157:H7 test has high sensitivity and specificity.

J Clin Microbiol, 2000 May, 38(5), 1786 - 90
Sequence diversity of the Escherichia coli H7 fliC genes: implication for a DNA-based typing scheme for E . coli O157:H7; Wang L et al.; Flagellar (H) antigens are mostly encoded by genes at the fliC locus in E . coli . We have sequenced 11 H7 fliC genes from Escherichia coli strains that belong to seven O serotypes . These sequences, together with those of nine other H7 fliC genes (from strains of three different O serotypes) sequenced recently (S . D . Reid, R . K . Selander, and T . S . Whittam, J . Bacteriol . 181:153-160, 1999), include 10 different sequences . The differences between these 10 sequences range from 0.06 to 3.12% . By comparison with other E . coli flagellin genes, we have identified primer length sequences specific for H7 genes in general and others specific for H7 genes of O157 and O55 strains: the specificity was confirmed by PCR testing the type strains for all 53 E . coli H types . We have previously identified genes specific for the E . coli O157 antigen, and use of the combination of O157- and H7-specific primers allows the sensitive and rapid detection of O157:H7 E . coli strains, which cause the majority of hemorrhagic colitis cases.

FEBS Lett, 2000 Apr 28, 472(2-3), 247 - 53
The C-A mismatch base pair and the single-strand terminus in the E . coli initiator tRNA(fMet) acceptor stem adopt unusual conformations; Zuleeg T et al.; Acceptor stem variants of tRNA(fMet) (Escherichia coli) have been characterized by nuclear magnetic resonance . The wild type contains a C1-A72 mismatch pair which is crucial for its biological function . For comparison, the mismatch was replaced by regular pairs U1-A72 and C1-G72 . Further variants contain an altered discriminator base, G73, or a G1-C72/U73 combination . The stems of variants U1-A72/A73 and C1-G72/A73 have A-RNA geometry, which extends essentially to the single-strand terminus . C1-A72/G73 variant and wild type are structurally almost identical . C1 and A72 adopt peculiar conformations with C1 being largely destacked with respect to G2, while A73 stacks upon C1 . The unique arrangement of the mismatch causes a distinctly different orientation of the single-strand terminus compared to variants with regular 1-72 base pairs, and to formyltransferase-complexed tRNA(fMet).

Biol Chem, 2000 Mar, 381(3), 183 - 92
Interaction of E . coli single-stranded DNA binding protein (SSB) with exonuclease I . The carboxy-terminus of SSB is the recognition site for the nuclease; Genschel J et al.; The 3'-5' single-stranded DNA(ssDNA) degrading exonuclease I of E . coli directly interacts with the E . coli ssDNA binding protein (EcoSSB) . Analytical ultracentrifugation shows that all 4 carboxy-termini of an EcoSSB tetramer bind exonuclease I . Binding is weakened by increasing salt concentrations, indicating the involvement of the negatively charged amino acids of the carboxy-terminus of SSB . Mutant SSB proteins EcoSSBP176S (ssb-113) and EcoSSBF177C do not bindtoexonuclease I while EcoSSBG15D (ssb-3) does bind . In a co-precipitation assay we show that the absence of the lastten amino acids (PMDFDDDIPF) completely abolishes binding of EcoSSB to exonuclease I . The interaction does not depend on the presence of the correct amino-terminal DNA binding domain or the amino acid sequences between the DNA binding domain and the last ten amino acids . A synthetic peptide (WMDFDDDIPF), corresponding to the last nine amino acids of EcoSSB, specifically inhibits the interaction . Both EcoSSBP176S and EcoSSBF177C SSBs bind DNA similar to wild-type EcoSSB, indicating that the phenotype of ssb-113 is not an indication of altered DNA binding . The repair deficiency of either ssb-3 or ssb-113 strain can be complemented by overexpression of the respective other mutant.

J Bacteriol, 2000 May, 182(10), 2741 - 5
Regulation of the Escherichia coli K5 capsule gene cluster: evidence for the roles of H-NS, BipA, and integration host factor in regulation of group 2 capsule gene clusters in pathogenic E . coli; Rowe S et al.; The expression of Escherichia coli group 2 capsules (K antigens) is temperature dependent, with capsules only being expressed at temperatures above 20 degrees C . Thermoregulation is at the level of transcription, with no detectable transcription at 20 degrees C . Using the E . coli K5 capsule gene cluster as a model system, we have shown that the nucleoid-associated protein H-NS plays a dual role in regulating transcription of group 2 capsule gene clusters at 37 and 20 degrees C . At 37 degrees C H-NS is required for maximal transcription of group 2 capsule gene clusters, whereas at 20 degrees C H-NS functions to repress transcription . The BipA protein, previously identified as a tyrosine-phosphorylated GTPase and essential for virulence in enteropathogenic E . coli, was shown to play a similar role to H-NS in regulating transcription at 37 and 20 degrees C . The binding of integration host factor (IHF) to the region 1 promoter was necessary to potentiate transcription at 37 degrees C and IHF binding demonstrated by bandshift assays . The IHF binding site was 3' to the site of transcription initiation, suggesting that sequences in the 5' end of the first gene (kpsF) in region 1 may play a role in regulating transcription from this promoter at 37 degrees C . Two additional cis-acting sequences, conserved in both the region 1 and 3 promoters, were identified, suggesting a role for these sequences in the coordinate regulation of transcription from these promoters . These results indicate that a complex regulatory network involving a number of global regulators exists for the control of expression of group 2 capsules in E . coli.

Radiats Biol Radioecol, 2000 Jan-Feb, 40(1), 10 - 4
{The nature of SOS response in E . coli K-12 (uvrA) cells exposed to the different ultraviolet doses}; Komova OV et al.; The kinetic and dose dependencies of the SOS-induction in E . coli (uvrA) cells exposed to UV light were investigated . Below 2 J/m2 the rate of the SOS-induction increased with dose . The maximal level of the SOS-response was proportional to the UV dose . Pyrimidine dimers were necessary for the induction . In the dose range 2-10 J/m2 the rate of the SOS-induction decreased with dose . The dose-response curve was non-linear . Pyrimidine dimers were not required for the induction . The nature of the molecular events leading to the SOS-induction at low and high doses was discussed.

Mol Cells, 2000 Feb 29, 10(1), 76 - 82
Transfer RNA identity change in anticodon variants of E . coli tRNA(Phe) in vivo; Kim HS et al.; The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases . We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E . coli tRNA(Phe) . Constructing different anticodon mutants of E . coli tRNA(Phe) by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA(Phe); the anticodons corresponded to 16 amino acids and an opal stop codon . To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E . coli strains containing these tRNA(Phe) genes in a medium which permitted tRNA induction . Fourteen mutant tRNA genes did not affect host viability . However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors . Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo . Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine . These three tRNAs and tRNA(Phe) are located in the same cluster in a sequence similarity dendrogram of total E . coli tRNAs . The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.

Mol Cells, 2000 Feb 29, 10(1), 8 - 12
Molecular cloning of a cDNA encoding ribosome inactivating protein from Amaranthus viridis and its expression in E . coli; Kwon SY et al.; In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis . To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs . Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced . It was 1,047 bp and contained an open reading frame encoding 270 amino acids . The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs . E . coli cells expressing A . viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A . viridis RIP presumably inactivated E . coli ribosomes . In addition, recombinant A . viridis RIP cDNA produced by E . coli had translation inhibition activity in vitro.

Biochim Biophys Acta, 2000 Apr 21, 1457(3), 211 - 28
Proton translocating nicotinamide nucleotide transhydrogenase from E . coli . Mechanism of action deduced from its structural and catalytic properties; Bizouarn T et al.; Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria . Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains . Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively . Domain II spans the membrane and constitutes at least partly the proton translocating pathway . Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented . The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP(+) by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site.

Arch Biochem Biophys, 2000 Apr 15, 376(2), 313 - 9
Chemical modification of SH groups of E . coli phosphofructokinase-2 induces subunit dissociation: monomers are inactive but preserve ligand binding properties; Guixe V; Modification of Escherichia coli phosphofructokinase-2 (Pfk-2) with N-(1-pyrenil)maleimide results in an enzyme form that is inactive . However, the rate of modification is drastically reduced in the presence of the allosteric effector MgATP . The stoichiometry of the label incorporation was found to be 2.03 +/- 0.035 mol of the reagent/mol of subunit, in agreement with the number of titratable SH groups by 5,5'-dithiobis(2-nitrobenzoic acid) in the labeled protein . HPLC gel filtration experiments demonstrate that native Pfk-2 is a dimer in the absence of ligands, while in the presence of MgATP a dimer-tetramer transition is promoted . In contrast, the modified enzyme eluted as a monomer and the presence of MgATP was not able to induce aggregation . Although the modified monomers are inactive, the dissociation constants for the substrates and the allosteric effector MgATP, measured by following the fluorescence of the binding probe, are the same as for the native enzyme . Quenching of pyrene fluorescence emission of labeled phosphofructokinase-2 monomers by acrylamide gave downward curved Stern-Volmer plots, with very similar quenching efficiencies for the control and for the fructose-6-P and MgATP-enzyme complexes . These results show the presence of SH groups in the interface of Pfk-2 subunits, critical for subunit interactions, and that conformational changes occurring through the dimers are essential for catalytic activity .

Acta Virol, 1999 Aug, 43(4), 205 - 11
C-terminal region of VP1 of selected foot-and-mouth disease virus serotypes: expression in E . coli and affinity purification; Ratish G et al.; Foot-and-mouth disease (FMD), one of the most contagious and economically important diseases of farm animals, is caused by a FMD virus (FMDV) which belongs to the family of Picornaviridae . The virus occurs as seven serotypes of which four (A, O, C and Asia 1) are prevalent in India . Immunoprophylaxis supported by precise diagnosis is the prime requirement for achieving the success in controlling the disease . Recently, recombinant DNA technology is gaining importance for the production of cost-effective and safer diagnostics and immunogens . Based on this approach, cDNA of a part of gene for major variable immunogenic region, VP1, of FMDV of four serotypes (A22, O, C and Asia 1) was amplified by PCR and cloned into expression vector . The expression of the 16 K protein gene from the clones was induced with IPTG and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and {35S}-methionine labeling . The immunoreactivity of the labeled proteins was assayed by immunoprecipitation with anti-FMDV type-specific sera . Since the proteins contain 6 His residues at the N-terminal end, their affinity purification was carried out using nickel nitrilo-tri-acetic acid (Ni-NTA) agarose matrix . The proteins were found to be immunoreactive and the useful in the FMD diagnosis.

Biochemistry, 2000 Apr 11, 39(14), 4068 - 74
Using a targeted chemical nuclease to elucidate conformational changes in the E . coli 30S ribosomal subunit; Muth GW et al.; Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem . The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous . This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation) . By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain . These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.

Hua Xi Yi Ke Da Xue Xue Bao, 1998 Dec, 29(4), 355 - 9
{Cloning, characterization and expression in E . coli of the flagellin gene from Leptospira interrogans Serovar lai}; You Z et al.; To obtain a large amount of recombinant endoflagellin protein of leptospsira, we have cloned the fla B gene from Leptospira interrogans serovar lai strain 017, which encodes the core flagellar protein . The entire open reading frame was amplified by polymerase chain reaction, which was digested with endonucleases Sal I and Cla I, then was orientationally cloned into the expression vector, pT7-7 . The recombinant plasmid was transformed into Escherichia coli JM109 (DE3) for expression . After induction with IPTG, a 34 kd expression protein was detected by SDS-PAGE . The amount of recombinant protein was estimated as 11.8% of the whole bacterial lysate proteins . Probed with insert fragments, Southern blot analysis indicated that possibly two gene related to fla B were present in the genome of Leptospira interrogans serovar lai stain 017 . Western blot analysis showed that the 34 kd expression protein reacting with rabbit polyclonal antiserum raised against leptospira endflagellin.

Science, 2000 Mar 31, 287(5462), 2497 - 500
Single-molecule study of transcriptional pausing and arrest by E . coli RNA polymerase; Davenport RJ et al.; Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coli RNA polymerase in real time over long template distances . These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop . The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state . The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes.

Science, 2000 Mar 31, 287(5462), 2482 - 6
Structure of the RNA polymerase domain of E . coli primase; Keck JL et al.; All cellular organisms use specialized RNA polymerases called "primases" to synthesize RNA primers for the initiation of DNA replication . The high-resolution crystal structure of a primase, comprising the catalytic core of the Escherichia coli DnaG protein, was determined . The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the "toprim" fold . On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase.

Biochemistry, 2000 Apr 4, 39(13), 3708 - 17
Kinetic and mechanistic analysis of the E . coli panE-encoded ketopantoate reductase; Zheng R et al.; Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway . The enzyme encoded by the panE gene from E . coli K12 was overexpressed and purified to homogeneity . The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da . The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP(+) release . The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism . An enzyme group exhibiting a pK value of 8.4 +/- 0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 +/- 0.2 serves as a general base in the direction of pantoate oxidation . The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by (1)H NMR spectroscopy . Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis . Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion . The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, DeltaG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation.

Wiad Lek, 1998, 51 Suppl 4, 233 - 6
{New possibilities of treatment with PEG-L-asparaginase in patients with acute lymphoblastic leukemia sensitized to l-asparaginase E.coli and erwinase}; Sikorska-Fic B et al.; L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia in children and adults . Use of L-aspa E . Coli as well as Erwinase is not possible in all cases because of the side effects, mainly allergic reactions and disfunction of pancreas . Recently, the new form of the enzyme PEG-L-asparaginase was introduced . Binding L-asparaginase E . coli to polyethylene glycol a decreased its toxicity, extended its plasma half-live, not significantly affecting the efficacy . The aim of the study was to examine the results of PEG-L-asparaginase administration in five children with acute lymphoblastic leukemia, and the symptoms of intolerance to L-aspa E . Coli or Erwinase . There were three children with newly diagnosed ALL and two children with first relapse of ALL, treated according to New York Protocol and BFM-90 Protocol for ALL relapses respectively . PEG-L-asparaginase (Oncaspar) was administered in the dose of 2500 IU/m2 . According to the protocol four children received 11 courses of treatment with the full dose of the drug . The number of doses for individual patient varied from one to six . The short-lived nettlerash was observed in one patient during two subsequent infusions of the drug . Hydrocortisone and antihistamine drugs were administered . Treatment with PEG-asparaginase was discontinued in one child, who developed dyspnea, nausea, vomiting and face rash during the third dose of the drug . Oncaspar is the valuable drug, which enabled continuation of treatment according to protocol in four out of five children with bad tolerance to routinely used L-asparaginase preparations.

J Biochem (Tokyo), 2000 Jan, 127(1), 1 - 7
Site-directed mutational analysis of DnaA protein, the initiator of chromosomal DNA replication in E . coli; Mizushima T; DnaA protein, the initiator for chromosomal DNA replication in Escherichia coli, has various activities, such as oligomerization (DnaA-DnaA interaction), ATP-binding, ATPase activity and membrane-binding . Site-directed mutational analyses have revealed not only the amino acid residues that are essential for these activities but also the functions of these activities . Following is a summary of the functions and regulatory mechanisms of DnaA protein in the initiation of chromosomal DNA replication . ATP-bound DnaA protein, but not other forms of the protein binds to the origin of DNA replication and forms oligomers to open-up the duplex DNA . This oligomerization is mediated by a DnaA-DnaA interaction through the N-terminal region of the protein . After initiation of DNA replication, the ATPase activity of DnaA protein is stimulated and DnaA protein is inactivated to the ADP-bound form to suppress the re-initiation of DNA replication . DnaA protein binds to acidic phospholipids through an ionic interaction between basic amino acid residues of the protein and acidic residues of phospholipids . This interaction seems to be involved in the re-activation of DnaA protein (from the ADP-bound form to the ATP-bound form) to initiate DNA replication after the appropriate interval.

J Mol Biol, 2000 Mar 31, 297(3), 803 - 8
Infrared dichroism of twisted beta-sheet barrels . The structure of E . coli outer membrane proteins; Marsh D; The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis . For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet . Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels . Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees . Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel .

Berl Munch Tierarztl Wochenschr, 2000 Feb, 113(2), 67 - 70
{Action of the vaccination of sows against E . coli infections with a subsequent postpartum booster}; Bilkei G et al.; In an eastern european pig productions unit with high prevalence of suckling piglets diarrhoea during late lactation the following trial was conducted: The sows were assigned to an experimental and to a control group and were treated as follows: Group one (15 sows) were vaccinated with a single 2 ml dose of Porcovac Plus (Hochst Roussel Vet.) during their late pregnancy (gilts were vaccinated twice) . Booster vaccination was performed between day 2-7 p.p . Group two (15 sows) were vaccinated during their late pregnancy the same was as the sows in the group one, but received no p.p . Booster . The following parameter were evaluated . A: Preweaning diarrhoea B: Preweaning mortality C: Three weeks weaning litter weights D: Postweaning mortality E: Average weight gain during the first 3 weeks postweaning The results revealed a marked difference between the groups (group 1: 16.1% vs . group 2: 23.3%) regarding preweaning diarrhoea (parameter A) . A similar difference was to be seen regarding parameter B (preweaning mortality) between the groups (group 1: 7.5% vs . group 2: 10.7%) . In spite of this there was a non significant difference as regards weaning litter weights (parameter C) between group 1: 59.2 +/- 2.4 kg and group 2: 57 +/- 2.2 kg . Postweaning parameters showed better results in the booster vaccinated group regarding both evaluated parameter as well . Regarding piglet mortality (parameter D) there was a marked difference between the group 1 (0.67%) and group 2 (2.1%) to be seen . Regarding average daily gain (parameter E) between group 1 (470 +/- 11 gr) and group 2 (380 +/- 9 gr) there was a significant difference (p < 0.05) to be discovered . It is the opinion of the authors that p.p . Booster vaccination of the sows is an economically important tool in large and small pig production units.

Cell, 2000 Mar 3, 100(5), 537 - 49
Solution structure of the E . coli 70S ribosome at 11.5 A resolution; Gabashvili IS et al.; Over 73,000 projections of the E . coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex . This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges . Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association . Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop . Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

Vaccine, 2000 Apr 14, 18(20), 2147 - 54
Immunoadjuvant activities of E . coli- and plasmid-expressed recombinant chicken IFN-alpha/beta, IFN-gamma and IL-1beta in 1-day- and 3-week-old chickens; Schijns VE et al.; In the present study we assessed the capacity of recombinant E . coli- or plasmid-expressed chicken interferons (IFN) and chicken IL-1beta, to exert immunostimulatory activities for humoral immune responses, in day-old and adult chickens . We observed that both recombinant E . coli-expressed chicken IFN-alpha/beta and IFN-gamma facilitated the induction of a primary and also a secondary antibody response, using tetanus toxoid (TT) as a bacterial model antigen, in immunologically mature 3-week-old chickens . In contrast, no improvement of antibody either type of chicken IFN was co-injected with inactivated Infectious Bursal Disease Virus (IBDV) antigen . TT-specific antibody formation was marginally increased by co-injection of recombinant E . coli-expressed chicken IL-1beta . Combined administration of IFN-alpha/beta plus IFN-gamma or IL-beta increased responses to TT in an additive, but not synergistic fashion . Remarkably, no augmentation of antibody responses specific for TT, nor IBDV, was noted in day-old birds, receiving IFN-alpha/beta or IFN-gamma as adjuvant . Also, intramuscular immunization of 3-week-old birds, using plasmids encoding IFN-alpha/beta together with TT protein antigen, significantly increased the speed and magnitude of TT-specific antibody responses . Plasmids encoding chicken IL-beta or IFN-gamma had a minimal or inhibitory effect, respectively . These data indicate a potential for chicken cytokines as immunoadjuvant for particular types of chicken vaccine antigens.

Indian J Exp Biol, 1999 Aug, 37(8), 787 - 92
Cloning and expression of a nitroaryl reductase gene from Streptomyces aminophilus strain MCMB411 in E . coli JM109 and Streptomyces lividans TK64; Dey S; A partial genomic library was prepared in E . coli JM109 using pBR322 as vector and 2.4 kb Sau 3A I chromosomal fragment, encoding a nitroaryl reductase (nbr A) gene, from Streptomyces aminophilus strain MCMB 411 . From the library, 2.4 kb fragment was recloned in E . coli JM109 and S . lividans TK64 using pUC18 and pIJ702 as vectors respectively . The recombinant plasmids pSD103 and pSD105 expressed the reductase gene and exported the enzyme in periplasmic space of E . coli and in cytoplasm of S . lividans TK64 . The proteins expressed by E . coli and S . lividans had the same molecular mass (70 kD) as that expressed by parent strain, which suggested that the enzyme was processed similarly by all strains . Activities of the enzymes cloned in E . coli JM109 and S . lividans TK64 containing recombinant plasmids pSD103 and pSD105 respectively were optimum at 30 degrees C and pH 9 and requirement of cofactors was same as that of the parent strain.

Toxicon, 2000 Aug, 38(8), 1077 - 86
Melittin-mediated release of {3H}-oleic acid from E . coli cells is dependent upon heat- and trypsin-sensitive factor(s) in human serum; Saini SS et al.; Synthetic melittin mediated the release of {3H}-oleic acid ({3H}-OA) or its acylated lipids from {3H}-OA-labeled E . coli cells exposed to human serum . This phenomenon was not observed in the absence of serum and was calcium independent . The addition of serum was not required for melittin-mediated lysis of erythrocytes, although lysis was greater in the presence of serum than in its absence (P<0.001) . Trypsin treatment of human serum reduced the melittin-mediated release of {3H}-OA/acylated lipids, and this effect was more pronounced upon boiling the serum (P<0.01) . A kinetic study showed that maximum release of {3H}-OA/acylated lipids occurred within 3-6 min . Thin layer chromatography (TLC) analysis showed the lipids to be phosphatidyl ethanolamine (PE), phosphatidylethanol (PEt) and phosphatidic acid (PA) . There was no detectable level of oleic acid (OA), diacylglycerol (DAG), phosphatidyl choline (PC) or phosphatidyl serine (PS) . These findings suggested that a trypsin and heat-sensitive enzyme/factor present in the serum had a role in melittin-mediated action . These findings further showed that melittin activated phospholipase D (PLD), without affecting phospholipase A(2) (PLA(2)) or phospholipase C (PLC) activity.

J Med Microbiol, 2000 Mar, 49(3), 253 - 60
Protective features of monoclonal antibodies to Escherichia coli during experimental infection of mice with homologous and heterologous serotypes of E . coli; Raponi G et al.; Murine monoclonal antibodies (MAbs) MT1F and ARM1-4, recognising proteins on the surface of untreated Escherichia coli O6:K-, protected 100% of mice challenged intraperitoneally with 2 x LD50 of the same strain . MAb MT1F protected 70% of animals challenged with 2 x LD50 of E . coli O111:B4, whereas ARM1-4 gave complete protection . Lower survival was observed in mice given either MAb and challenged with E . coli O128:K-, with values ranging from 30 to 42% . However, the protection afforded against E . coli O111:B4 and E . coli O128:K- was significantly improved when the mice were pre-treated with a mixture of the two MAbs . Control mice, pre-treated with unrelated ascitic fluid and challenged with any of the E . coli serotypes, showed 100% mortality and organ histological lesions resembling those of the early stages of septic shock . The mice had high levels of circulating endotoxin and tumour necrosis factor-alpha (TNF-alpha) at 90 min after challenge . In contrast, mice treated with MAbs and surviving the infection displayed moderate histological lesions, enhanced bacterial clearance and lower serum levels of TNF-alpha, despite circulating endotoxin levels that were higher than in the control group . Protection by the MAbs was probably due to the prevention of the bacterial spread to organs and of the cascade of events leading to septic shock . This occurred in spite of the presence of high levels of circulating endotoxin.

Bioinformatics, 1999 Oct, 15(10), 860 - 1
Completing the E . coli proteome: a database of gene products characterised since the completion of the genome sequence; Thomas GH; A database collating research on E . coli genes whose products have been characterised subsequent to in silico predictions from the completed genome sequence.

Biophys J, 2000 Mar, 78(3), 1400 - 12
Interaction of phosphatidylserine synthase from E . coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies; Salamon Z et al.; The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy . The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films . Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-{phospho-rac-(1-glycerol)} (POPG) (75:25, mol/mol) . Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy . PS synthase exhibits a biphasic interaction with the bilayers . The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules . The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface . Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs . The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E . coli membranes.

FEMS Microbiol Lett, 2000 Mar 1, 184(1), 91 - 4
Human milk fractions inhibit the adherence of diffusely adherent Escherichia coli (DAEC) and enteroaggregative E . coli (EAEC) to HeLa cells; Nascimento de Araujo A et al.; Binding to a specific receptor is an essential step for most enteropathogens to initiate an intestinal infection . We analyzed the inhibitory effect of human milk and its protein components on adhesion of two diarrheagenic Escherichia coli strains, diffusely adherent E . coli (DAEC) and enteroaggregative E . coli (EAEC), to HeLa cells . Defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions, in concentrations lower than usually found in whole milk, inhibited both DAEC and EAEC adhesion, indicating that human milk components may contribute to the defense of the infants against enteropathogens.

Hua Xi Yi Ke Da Xue Xue Bao, 1998 Jun, 29(2), 127 - 31
{Cloning and orientation of cefoperazone resistance gene on plasmid pFC in E . coli HX88108}; Liang R et al.; Cefoperazone resistance gene (CPZr) has been cloned from plasmid pFC of E . coli HX88108 using the vector pMB9 (TCr, 5.3 kb) . The plasmid pFC DNA was partially digested with Sau3A I, and its 1-2 kb fragments were ligated into BamH I site of vector plasmid pMB9 . The recombinant DNA was then transformed into E . coli DH5 prepared using calcium chloride . CPZ resistant bacterial colonies were selected on the agar SOB plates containing CPZ (40 micrograms/ml) . The resistance to CPZ could be stably reserved in generation after generation . The recombinant plasmids which encoded CPZ resistance were designated pFL11, pFL25, pFL33, pFL82, pFL86 and pFL102 . Rapid small-scale preparation of plasmid and DNA restrication enzyme analysis were used for identification of bacterial colonies . Five plasmids DNA physical maps have been established . Comparison of recombinant plasmids maps with pFC map confirmed that the CPZr gene was oriented between nucleotide no . 3200 bp and no . 4800 + 40 bp of plasmid pFC total sequence . Its molecular weight was about 1.6 kb . There were EcoR I, Sma I and Pvu II sites within CPZr gene.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 143 - 50
Three-step chromatographic purification procedure for the production of a his-tag recombinant kinesin overexpressed in E . coli; Gibert S et al.; A kinesin gene has been cloned by RT-PCR (reverse transcription polymerase chain reaction) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine-tag) kinesin in E . coli in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography . Starting from several liters of culture, an ultrasonic homogenizer was used for cell disruption and an unclarified feedstock was obtained . From this homogenate, a protein was then purified by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology (Streamline chelating) . For this capture step, 100% of the recombinant protein was purified with more than 90% of purity . This step was followed by ion-exchange chromatography (Q Sepharose Fast Flow) for intermediate purification (96% purity, 53% recovery) and by size-exclusion chromatography with Superdex 75 as a polishing step (99% purity, 93% recovery) . We then separated two forms of kinesin, a dimer (70%) and a monomer (30%) . It was then possible to purify His-tag recombinant protein directly from feedstock in a rapid and efficient way and to isolate two forms of kinesin.

Nucleic Acids Res, 2000 Mar 1, 28(5), 1139 - 44
Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence; Yehudai-Resheff S et al.; Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway . The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I) . In order to understand the molecular mechanism of RNA poly-adenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme . Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA . PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G) . The 3'-ends of most of the mRNA molecules in bacteria are characterized by a stem-loop structure . We show here that in vitro PAP I activity is inhibited by a stem-loop structure . A tail of two to six nucleo-tides located 3' to the stem-loop structure is sufficient to overcome this inhibition . These results suggest that the stem-loop structure located in most of the mRNA 3'-ends may function as an inhibitor of poly-adenylation and degradation of the corresponding RNA molecule . However, RNA 3'-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.

Lett Appl Microbiol, 1999 Dec, 29(6), 434 - 6
Evaluation of a latex agglutination kit for the detection of human antibodies to the lipopolysaccharide of Escherichia coli O157, following infection with verocytotoxin-producing E . coli O157; Chart H; A total of 150 human sera was used to evaluate a commercial latex agglutination kit for detecting antibodies to the lipopolysaccharide of Escherichia coli O157 . A comparison of the kit with SDS-PAGE and immunoblotting showed that the kit had a sensitivity of 94.12%, a specificity of 99.15%, a positive predictive value of 96.97% and a negative predictive value of 99.15%.

Adv Exp Med Biol, 1999, 473, 155 - 61
Edema disease as a model for systemic disease induced by Shiga toxin-producing E . coli; Cornick NA et al.; Edema disease (ED) is a naturally occurring disease of weaned pigs caused by host adapted strains of E . coli that produce Shiga toxin (STEC) . We determined the temporal and quantitative relationships between intestinal colonization by STEC, levels of Shiga toxin (Stx2e) in the gut, in the blood, and clinical manifestations of ED . Bacterial colonization (10(8) CFU/cm ileum) was highest 4 days post inoculation (pi) in animals that did not develop clinical disease and 6 days pi in animals with clinical signs of ED . The mean time for the development of clinical signs of ED was 6 days pi (range 4-10) . Average peak titers of Stx2e in the ileum were 1:16,384 in asymptomatic animals and 1:32,768 in clinical animals . Titers of Stx2e in the feces reflected the toxin titers in the ileum but were lower . Intestinal titers of Stx2e and the density of bacterial colonization were predictive of clinical ED for a group of animals but not for individuals . Approximately 50% of the pigs that had Stx2e titers of > or = 1:4096 and a bacterial density of > or = 10(6) CFU/cm in their ileum, had clinical ED . Pigs that had intestinal Stx2e titers < 1:4096 were asymptomatic . Stx2e was detected in the red cell fraction of blood from some of the pigs with clinical ED and in some that were asymptomatic . Stx2e was not detected in the serum of any animals . ED may be a useful model for predicting the temporal and quantitative relationships between bacterial colonization, Stx levels in the gut and blood and systemic disease for STEC in other species.

J Mol Biol, 2000 Feb 11, 296(1), 155 - 68
Crystal structure at 2.4 A resolution of E . coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate; Scarsdale JN et al.; Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor . Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor . Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme . We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 % . This structure reveals the interactions of both cofactors and glycine substrate with the enzyme . Comparison with the E . coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site . Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate . The tetrameric quaternary structure of liganded E . coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes . SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E . coli enzymes . The structure of the E . coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants .

Biotechniques, 2000 Jan, 28(1), 90 - 4
Novel kanamycin/neomycin phosphotransferase cassette increases transformation efficiency in E . coli; Rashid MB et al.; Stable transformation depends on the efficient delivery of DNA into cells and the robust expression of genes that encode proteins which provide resistance to selective (cytotoxic) compounds . We have examined the possibility that altering the 5'untranslated region (UTR) of a selectable marker may increase transformation efficiency . A 15-nucleotide synthetic UTR (the so-called universal translational enhancer {UTE}) was placed upstream of a kanamycin/neomycin phosphotransferase (kanaR) gene to create a novel expression cassette, UTE-kanaR . In comparison to a wild-type version of kanaR, UTE-kanaR produced up to 30-fold more transformants in E . coli . The superior performance of UTE-kanaR was independent of the promoter strength, indicating that the gene may find general use in routine transformation experiments.

Biotechniques, 2000 Jan, 28(1), 76 - 82
Detection of eukaryotic cDNA in differential display is enhanced by the addition of E . coli RNA; Melichar H et al.; We describe a method to enhance the sensitivity of eukaryotic cDNA detection in differential display (DD) . Typically, DD protocols require between 200 and 500 ng RNA for each reverse transcription reaction . The addition of Escherichia coli RNA before reverse transcription of eukaryotic RNA increases the detection of DD patterns more than tenfold . The method broadens the applicability of DD and allows the identification of genes that are differentially expressed when the amount of eukaryotic RNA is limited.

Protein Expr Purif, 2000 Feb, 18(1), 11 - 9
In vitro refolding of heterodimeric CapZ expressed in E . coli as inclusion body protein; Remmert K et al.; CapZ is a heterodimeric Ca(2+)-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells . It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length . Here we report the expression of the two muscle-specific isoforms alpha2 and beta1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies . Optimal renaturation conditions were obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DTT, 1 mM PMSF, and 100 mM Tris, pH 7.4 . The refolding mixture was incubated for 24 h at 15 degrees C and the protein was concentrated by ultrafiltration . Biochemical characterization of the recombinant heterodimer revealed actin binding activities indistinguishable from those of native CapZ as purified from chicken skeletal muscle . Using the same protocol, we were able to refold the beta1, but not the alpha2 isoform as a single polypeptide, indicating a role for beta1 as a molecular template for the folding of alpha2 . The reported recombinant approach leads to high yields of active heterodimer and allows the renaturation and characterization of the beta subunit .

Indian J Exp Biol, 1999 Jun, 37(6), 536 - 40
Comparative studies on immunoreactivity of truncated recombinant proteins of foot and mouth disease virus (FMDV) produced in E.coli and insect cells; Viswanathan S et al.; For effective FMD control programme, India needs large quantities of cheaper diagnostics in addition to vaccine . Diagnostic reagents produced through conventional methods may not be able to meet such requirements . Alternatively, rDNA technology using suitable heterologous systems that permit production of recombinant antigens to the most native form may be exploited . Studies conducted in our laboratory have led us to select carboxy terminal part of VP1 for expression and evaluation . The protein, which was purified from E.coli under denaturing conditions, was renatured and its reactivity was compared with the protein expressed in insect cells through recombinant baculovirus . The expressed protein in the insect cell whole lysate reacted more efficiently with antibodies raised against whole virus than the purified and renatured protein produced in E.coli . But for its lower reactivity, protein produced from E.coli was found to be suitable in type detection . In addition, the size of the protein is small (16 kD) and production and purification of it from E.coli may be cost effective . Hence, it may be exploited for FMDV typing.

J Theor Biol, 2000 Jan 21, 202(2), 175 - 85
Ribosome traffic in E . coli and regulation of gene expression; Lesnik T et al.; The ribosome traffic during translation of E . coli coding sequences was simulated, assuming that the rate of translation of individual codons is limited by the cognate tRNA availability . Actual translation rates were taken from Solomovici et al . (J . theor . Biol . 185, 511-521, 1997) . The mean translation rates of the 4271 sequences cover a broad, two-fold range, whereas the local rate of translation along messengers varies three-fold on average . The simulation allows one to sketch the ribosome traffic on the polysome, in particular by providing the extent of mRNA sequences uncovered between consecutive ribosomes and the time during which these sequences are exposed . These parameters may participate in the control of mRNA stability and transcriptional polarity . By averaging the translation rates in a 17-codon window, assumed to be the sequence covered by a translating ribosome, and sliding this window along a given coding sequence, the addresses KMAX and KMIN, and the times TMAX and TMIN of respectively the slowest and the fastest translated window were determined . It is shown that under the assumptions made, TMAX sets the number of proteins translated from a given mRNA molecule per unit time, in case the delay between consecutive translation starts is below TMAX . Both windows display two strong biases, one as expected on the usage of codon frequencies, and the other surprisingly on the occurrence of amino acids .

J Physiol Pharmacol, 1999 Dec, 50(4), 551 - 65
Biphasic response to lipopolysaccharide from E . coli in the isolated ventilated blood-perfused rat lung; Chlopicki S et al.; We characterised early circulatory and respiratory responses to lipopolysaccharide from E . coli (LPS, serotype 0127:B8) in the isolated, ventilated and perfused rat lung preparation . Lungs were isolated from anaesthetised Wistar rats and perfused with full blood, platelet rich plasma (PRP), platelet poor plasma (PPP) or Krebs-Henseleit solution (KH) . LPS (300 microg/ml) injected into the blood-perfused lung induced a characteristic biphasic response consisting of an immediate, transient decrease in respiratory tidal volume and an increase in pulmonary perfusion pressures followed by a delayed decrease in respiratory tidal volume . An immediate respiratory/circulatory response to LPS was of considerable magnitude only in full blood-perfused lung whereas the delayed response was fully expressed irrespective whether blood, PRP, PPP or KH was used for the lung perfusion . Immediate respiratory/circulatory response was inhibited by WEB 2170 (100 microM), a PAF receptor antagonist, and by camonagrel (300 microM), a TXA2 synthase inhibitor, but not by MK 571 (100 microM), a cysteinyl leukotriene receptor antagonist . Delayed respiratory response was inhibited by camonagrel only . In summary, we demonstrated that the immediate coupled respiratory/circulatory response is mediated by blood cell-derived PAF and TXA2 whereas the delayed uncoupled respiratory response is mediated by lung parenchyma-derived TXA2.

Biochemistry, 2000 Jan 18, 39(2), 356 - 61
A conformational change in E . coli DNA polymerase I (Klenow fragment) is induced in the presence of a dNTP complementary to the template base in the active site; Dzantiev L et al.; It is well established that the insertion of a nucleotide into a growing DNA chain requires a conformational change in the structure of a DNA polymerase . These enzymes have been shown to bind a primer-template in the open conformation and then upon binding of a complementary dNTP undergo a conformational rearrangement to the closed ternary complex . This movement results in the positioning of the incoming nucleotide in the proper geometry for the nucleophilic attack by the 3'-hydroxyl of the primer . In this work, tryptic digestion experiments were performed to detect this conformational change in the structure of the exonuclease-deficient DNA polymerase I (Klenow fragment) . Three distinct digestion patterns were observed: one for the polymerase alone, one for the binary complex with the primer-template, and one for the ternary polymerase-DNA-dNTP complex . The latter conformational change leads to a stable ternary closed complex formation only when the correct nucleotide is present in the reaction mixture . Positioning of nucleotides with incorrect geometry in the protein active site inhibits or eliminates formation of the closed complex . Similarly, this conformational change is inhibited when the primer terminus of the DNA molecule is altered by the presence of the 2'-hydroxyl.

Radiat Res, 2000 Feb, 153(2), 181 - 5
Helium-neon laser preirradiation induces protection against UVC radiation in wild-type E . coli strain K12AB1157; Kohli R et al.; We have observed that preirradiation with a helium-neon laser (632.8 nm) induces protection against UVC radiation in wild-type E . coli strain K12AB1157 . The magnitude of protection was found to depend on the helium-neon laser irradiance, exposure time, and period of incubation between helium-neon laser exposure and subsequent UVC irradiation . The optimum values for dose, irradiance and interval between the two exposures were found to be 7 kJ/m(2), 100 W/m(2) and 1 h, respectively . The possible involvement of singlet oxygen in the helium-neon laser-induced protection is also discussed.

Brain Res, 1999 Dec 11, 850(1-2), 47 - 54
Neuron-specific expression of reporter gene in transgenic mice carrying the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha 1A subunit gene fused to E . coli lacZ reporter gene; Takahashi E et al.; To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q type calcium channel alpha 1A subunit gene, transgenic mice carrying a 0.5-kb, 1.5-kb, 3.0-kb or 6.3-kb 5'-upstream region of the gene fused to Escherichia coli lacZ reporter gene were produced . In transgenic mice carrying the 1.5-kb, 3.0-kb or 6.3-kb 5'-upstream region, the reporter gene was exclusively expressed in the nervous system, although those with the 0.5-kb 5'-upstream region failed to show reporter expression . Histological examinations showed that the three 5'-upstream regions induced distinct expression patterns of the reporter gene in the CNS and adrenal medulla . The 1.5-kb 5'-upstream region drove reporter gene expression in the olfactory bulb, dorsal cortex and hippocampus, while the regulatory element for the expression in the amygdaloid nucleus, septum, habenula medial nucleus, choroid plexus, substantia nigra, inferior colliculus, pontine nucleus and cerebellum was located in the 5'-upstream sequence between 1.5 kb and 6.3 kb . In the cerebellum, the expression of the reporter gene was induced by the 3.0-kb region in granule cells, whereas it was induced by the 6.3-kb region in Purkinje cells . The expression of the reporter gene in chromaffin cells in the adrenal medulla was induced only by the 6.3-kb 5'-upstream region . These results suggest that the expression of the mouse P/Q-type Ca2+ channel alpha 1A subunit gene is regulated in a complex fashion by both positive and negative cis-regulatory elements.

Biochim Biophys Acta, 2000 Jan 3, 1476(1), 109 - 28
Formycin A and its N-methyl analogues, specific inhibitors of E . coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-->ligand fluorescence resonance energy transfer; Kierdaszuk B et al.; Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz . formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)) . Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i) . Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i) . Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching . With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive . Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands . With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i) . Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM) . The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model . Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA . For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer . Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes (<tau>) affected by their interactions . Complex formation resulted in a 2-fold increase in <tau> of FA, and a 2-fold decrease in <tau> of enzyme fluorescence . The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species . The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.

Eur J Hum Genet, 1999 Dec, 7(8), 884 - 8
Carbohydrate-deficient glycoprotein syndrome type 1A: expression and characterisation of wild type and mutant PMM2 in E . coli; Kjaergaard S et al.; We have identified the PMM2 genotypes of 22 unrelated Danish patients with carbohydrate-deficient glycoprotein syndrome type 1A: R141H/F119L (18), R141H/C192G (1), F119L/F119L (1), F119L/G117R (1) and D223E/T237R (1) . The lack of patients homozygous for R141H is statistically highly significant, but unexplained . In order to investigate the effect of PMM2 mutations on phosphomannomutase (PMM2) activity, PMM2-cDNA was cloned into a pET3a vector . Following introduction of mutations into PMM2-cDNA by site-specific mutagenesis, wild type and mutant PMM2-cDNA were expressed in E . coli Bl21(DE3) cells, and the activity of PMM2 was determined by an enzymatic assay using mannose 1-phosphate as substrate . Recombinant R141H, G117R, and T237R PMM2 had no detectable catalytic activity, and the F119L PMM2 had 25% of the activity of the wild type . The activity of the C192G and D223E PMM2 was in the normal range, but the affinity for their substrate was lower, and the proteins were more sensitive to increased temperatures . Each patient has at least one mutation which retains residual PMM2 activity . Our results support the hypotheses that a genotype conveying residual PMM2 catalytic activity is required for survival, and that homozygosity for R141H impairs PMM2 to a degree incompatible with life.

J Virol Methods, 1999 Dec, 83(1-2), 83 - 9
Characterization of monoclonal antibodies against bovine herpesvirus 1 gD fusion protein expressed in E . coli; Lyaku JR et al.; A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E . coli expressed bovine herpesvirus-1 (BHV-1) gD fusion protein were produced following the fusion of Sp2/0 myeloma cells with splenocytes from BALB/c mice immunized previously with immunoaffinity purified BHV-1 gD fusion protein . An indirect fluorescent antibody test (IFAT) using BHV-1 infected MDBK cells was used for the selection of positive hybridomas secreting specific antibody . The monoclonal antibody isotypes were 11 IgM, six IgG2b, one IgG1 and two IgG3 . All MAbs reacted positively with the E . coli expressed BHV-1 gD fusion protein, BHV-1 infected MDBK cell lysates and PCR BHV-1 gD transcription-translation polypeptide antigens by an ELISA.

J Biotechnol, 1999 Oct 8, 75(2-3), 241 - 50
The use of elements of the E . coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations; Schroeckh V et al.; The inducible glnA promoter 2 of the E . coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities . It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM . This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system . These artificial constructions result in enhanced recombinant gene expression in the production phase . Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control . A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene . The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half . It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2 . The optimized expression systems were successfully applied in low and high cell density cultivations . In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.

Artif Cells Blood Substit Immobil Biotechnol, 1999 Sep-Nov, 27(5-6), 475 - 81
Artificial cell microcapsules containing genetically engineered E . coli DH5 cells for in-vitro lowering of plasma potassium, phosphate, magnesium, sodium, chloride, uric acid, cholesterol, and creatinine: a preliminary report; Prakash S et al.; Lowering of plasma Mg, P, Na, Cl, uric acid, cholesterol, and creatinine is required in renal failure and other diseases . In this preliminary report, we studied the ability of artificial cells microencapsulated genetically engineered E . coli DH5 cells in lower K, Mg, P, Na, Cl, uric acid, cholestrol, creatinine, and billirubin from plasma in-vitro . Result shows that this novel approach has the ability to significantly lower these metabolites from the plasma in-vitro.

Biotechnol Bioeng, 2000 Jan 20, 67(2), 206 - 16
Facilitated downstream processing of a histidine-tagged protein from unclarified E . coli homogenates using immobilized metal affinity expanded-bed adsorption; Clemmitt RH et al.; The facilitated downstream processing of an intracellular, polyhistidine-tagged protein, glutathione S-transferase {GST-(His)(6)}, direct from unclarified E . coli homogenates using expanded beds of STREAMLINE chelating, has been investigated . A series of pilot experiments were used to develop preparative-scale separations of GST-(His)(6), initially in packed and then in expanded beds . Packed beds of Ni(2+)-loaded STREAMLINE chelating proved to have the highest 5% dynamic capacity for GST-(His)(6), of 357 U mL(-1) (36 mg mL(-1)) . When using immobilized Cu(2+) or Zn(2+), metal ion transfer was observed from the iminodiacetate ligands to the high-affinity chelator, GST-(His)(6) . The subsequent metal affinity precipitation of this homodimer resulted in operational problems . An equilibrium adsorption isotherm demonstrated the high affinity of GST-(His)(6) for immobilized Ni(2+), with a q(m) of 695 U mL(-1) (70 mg mL(-1)) and a K(d) of 0.089 U mL(-1) (0.0089 mg mL(-1)) . Ni(2+)-loaded STREAMLINE chelating was therefore selected to purify GST-(His)(6) from unclarified E . coli homogenate, resulting in an eluted yield of 80% and a 3.34-fold purification . The high dynamic capacity in the expanded mode of 357 U mL(-1) (36 mg mL(-1)) demonstrates that this specific interaction was not affected by the presence of E . coli cell debris .

Acta Chir Belg, 1999 Oct, 99(5), 226 - 9
The effect of pneumoperitoneum on bacterial clearance and RES functions in a model of E . coli peritonitis; Paksoy M et al.; The use of laparoscopic surgery in peritonitis has increased rapidly . The present study examined the effects of pneumoperitoneum on bacterial clearance . Spraque-Dawley rats were divided into six groups of seven animals . In groups 1 and 4, laparotomy with a midline incision was performed and 10(9) E . coli in a volume of 1 ml inserted into the peritoneal cavity . Groups 2, 3, 5, 6 received an identical quantity of E . coli by intraperitoneal injection . Groups 3 and 6 received carbon dioxide pneumoperitoneum at a constant pressure of 5 mmHg for 60 minutes after intraperitoneal injection of E . coli . In one hour groups; the mean bacterial counts per lung from the E . coli injection with laparotomy group was significantly higher than for the E . coli injection with pneumoperitoneum group (p < 0.05) . The mean bacterial counts per kidney in the E . coli injection with laparotomy group was higher compared with the E . coli injection and E . coli injection with pneumoperitoneum groups (p < 0.0001) . There was statistically significant difference in quantitative bacteraemia between the E . coli injection with laparotomy group and the E . coli injection or E . coli injection with pneumoperitoneum groups (p < 0.05) . In four-hour groups; the mean bacterial counts of lungs and liver-spleen were significantly higher in the E . coli injection with laparotomy group than in the E . coli injection and E . coli injection with pneumoperitoneum groups (p < 0.05 and p < 0.001 respectively) . The quantitative bacteria was significantly higher in the E . coli injection with laparotomy group than in the E . coli injection and E . coli injection with pneumoperitoneum groups (p < 0.05) . This study demonstrates that pneumoperitoneum impairs the clearance of bacteria from the peritoneal cavity in an experimental model of peritonitis . However, we could not detect the deleterious effects of pneumoperitoneum compared with laparotomy.

Bioorg Khim, 1999 Aug, 25(8), 638 - 40
{Mass spectrometry MALDI TOF for rapid qualitative evaluation of recombinant proteins production in E . coli}; Serebriakova MV et al.; The possible use of MALDI TOF MS for the analysis of Escherichia coli strains producing recombinant proteins was studied . It was shown that the target chimerical proteins might be rapidly detected in the strains.

Biochimie, 1999 Aug-Sep, 81(8-9), 901 - 7
An assessment of the role of intracellular free Ca2+ in E . coli; Holland IB et al.; We have previously proposed that fluctuations in Ca(2+) levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J . Theor . Biol . 134 (1998) 341-350) . This proposal implied the presence of Ca(2+) uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca(2+)-homeostasis, and perturbation of cell cycle processes when cellular Ca(2+) levels are depleted . We review the properties of new cell cycle mutants in E . coli and B . subtilis resistant to inhibitors of calmodulin, PKC or Ca(2+)-channels; the evidence for Ca(2+)-binding proteins including Acp and FtsZ; and Ca(2+)-transporters . In addition, the effects of EGTA and verapamil (a Ca(2+) channel inhibitor) on growth, protein synthesis and cell cycle events in E . coli are described . We also describe new measurements of free Ca(2+)-levels, using aequorin, in E . coli . Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature . Several of the mutants were also hypersensitive to EGTA and or Ca(2+) . However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase . The results also indicate that the expression of several genes in E . coli may be regulated by Ca(2+) . Cell division in particular appears very sensitive to the level of cell Ca(2+), with the frequency of division clearly reduced by EGTA and by verapamil . However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca(2+) including free Ca(2+), this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins . Measurement of free Ca(2+) in living cells indicated levels of 200-300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La(2+)-sensitive PHB-polyphosphate complexes involved in Ca(2+) influx . The evidence reviewed increasingly supports a role for Ca(2+) in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established.

Biochimie, 1999 Aug-Sep, 81(8-9), 841 - 6
Replication cycle dependent association of SeqA to the outer membrane fraction of E . coli; d'Alencon E et al.; The hemimethylated oriC binding activity of the E . coli heavy density membrane fraction (outer membrane) was investigated by DNase I footprinting experiments using membranes obtained from different replication stages of PC-2 (dnaCts) cells . The maximal binding activity was found at the beginning of replication cycle and then decreased gradually . The same pattern of variation was observed with SeqA protein detected in the membranes by immunoblotting . Both binding activity and the presence of SeqA were conserved in the outer membrane even after floating centrifugation of the heavy density membrane fraction in a sucrose gradient, indicating that SeqA in fact can associate with the membrane and that this association varies according to replication cycle . Site specific binding to hemimethylated oriC, of the heavy density membrane obtained from seqA mutant, could be restored by addition of a low amount of His-tagged SeqA protein.

Int J Food Microbiol, 1999 Nov 1, 52(1-2), 85 - 95
PCR assay for detection of the E . coli O157:H7 eae-gene and effect of the sample preparation method on PCR detection of heat-killed E . coli O157:H7 in ground beef; Uyttendaele M et al.; A PCR assay targeting the 3' end of the eae-gene of E . coli O157:H7 has been developed . It was shown to be specific for the E . coli O157:H7 eae-gene . Sensitivity of the PCR assay was 1 pg DNA or 10(3) cfu per PCR reaction . Subsequently, a study was conducted to examine the effect of the food matrix and the sample preparation method on PCR detection of non-viable cells using heat-killed E . coli O157:H7 in ground beef as a model system . Inoculated ground beef samples were subjected to either selective enrichment or immediately prepared for PCR analysis . Four sample preparation methods were applied: centrifugation, buoyant density centrifugation (BDC), immunomagnetic separation (IMS), and chelex extraction . Production of false-positive results due to amplification of the DNA of dead cells occurred if the number of heat-killed cells exceeded 10(8) cfu/g . For inocula <10(8) cfu/g, PCR results for heat- killed E . coli O157:H7 cells depended upon the sample preparation method . It was shown that the inclusion of two washings steps of the bacterial pellet before DNA extraction was the critical factor for elimination of false-positive results due to heat-killed cells in the centrifugation and BDC procedure . IMS did not produce false-positive results due to heat-killed cells (two washing steps were included in the IMS procedure) . With the chelex-extraction method, false-positive results due to heat-killed cells were obtained . An effect of the ground beef food matrix on the presence of amplifiable DNA was noted.

Bioorg Med Chem Lett, 1999 Oct 18, 9(20), 2959 - 62
Inhibition of E . coli growth by fullerene derivatives and inhibition mechanism; Mashino T et al.; Cationic ammonium fullerene derivatives (C60-bis(N, N-dimethylpyrrolidinium iodide) and C60-bis(N-methylpiperazinium iodide)) suppressed E . coli growth, whereas an anionic derivative (C60-dimalonic acid) did not . Both cationic derivatives inhibited E . coli dioxygen consumption . Inhibition of energy metabolism is concluded to be a mechanism of the growth inhibition effect of fullerene derivatives.

Bioorg Khim, 1999 Jul, 25(7), 548 - 53
{The dependence of the E.coli gene expression level on the structure of the translation initiation region (TIR) . IV . Distal complementary TIR interactions with the mRNA coding region}; Esipov RS et al.; To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA . A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation . The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.

FEBS Lett, 1999 Nov 12, 461(1-2), 6 - 8
The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to E . coli-based protein production; Matsuo E et al.; OmpT, an outer membrane-localized protease of Escherichia coli, cleaves a number of exogenous and endogenous proteins during their purification . SecY, an endogenous membrane protein, is a target of this artificial proteolysis in vitro . Here we report that SecY cleavage occurs even in cell extracts from ompT-disrupted cells, if they carry an F plasmid derivative . A gene, ompP, on the F plasmid was shown to be responsible for this proteolysis . These results indicate that the absence of an F-like plasmid should be checked when choosing a host strain for E . coli-based protein production.

Protein Sci, 1999 Oct, 8(10), 2065 - 71
Outer membrane protein A of E . coli folds into detergent micelles, but not in the presence of monomeric detergent; Kleinschmidt JH et al.; Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil . OmpA refolds upon urea dilution in the presence of certain detergents or lipids . To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles . OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation . Details of the chemical structure of the polar headgroup were unimportant for folding . The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series . Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding . Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.

Gene, 1999 Nov 1, 239(2), 351 - 9
Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli-H . pylori rpoD gene in E . coli; Shirai M et al.; We constructed and analyzed hybrid Escherichia coli-Helicobacter pylori rpoD genes in an E . coli rpoD mutant . It turned out that a hybrid consisting of E . coli rpoD with subdomain 4.2 of H . pylori rpoD (for -35 recognition) was functional . On the other hand, hybrids consisting of E . coli rpoD with domain 2 and the adjacent sequence of H . pylori rpoD (for core enzyme binding and -10 recognition) were non-functional . Intriguingly, a hybrid rpoD containing H . pylori subdomain 4.2 conferred higher activity for the H . pylori PureA as determined by xylE expression of PureA-xylE fusions, although the activity of the hybrid rpoD for the tac promoter was comparable to that of E . coli rpoD . The tsp of ureA in E . coli with the hybrid rpoD and E . coli rpoD were 15 and 17bp upstream from that in H . pylori, respectively . The comparison of PureA sequences in both E . coli and H . pylori indicated the existence of a -10 consensus sequence but little conservation of -35 sequences . Instead, the PureA in both H . pylori and E . coli contained an identical heptamer, GTTAATA, in the extended -35 region.

Microb Pathog, 1999 Nov, 27(5), 289 - 301
Phosphatidylethanolamine recognition promotes enteropathogenic E . coli and enterohemorrhagic E . coli host cell attachment; Barnett Foster D et al.; Using both solid phase and liposome aggregation assays, we screened a variety of glycolipids and phospholipids and found that EHEC and EPEC bind specifically and in a dose-dependent manner to PE . This binding was consistently observed whether the lipid was immobilized on a thin layer chromatography plate, in a microtitre well or incorporated into a unilamellar vesicle suspended in aqueous solution . There was no evidence of binding to other phospholipids such as phosphatidylcholine (PC) or phosphatidylserine (PS) . Bacterial binding to two epithelial cell lines also correlated with the level of outer leaflet PE and was reduced following preincubation with anti-PE . The PE-binding phenotype of EPEC appeared to correlate with the bundle-forming pilus (bfp) genotype of a number of clinical isolates . These results provide evidence of a receptor role for PE in the adhesion of EHEC and EPEC to host cells .

J Mol Biol, 1999 Nov 5, 293(4), 815 - 34
An oligomeric form of E . coli UvrD is required for optimal helicase activity; Ali JA et al.; Pre-steady-state chemical quenched-flow techniques were used to study DNA unwinding catalyzed by Escherichia coli UvrD helicase (helicase II), a member of the SF1 helicase superfamily . Single turnover experiments, with respect to unwinding of a DNA oligonucleotide, were used to examine the DNA substrate and UvrD concentration requirements for rapid DNA unwinding by pre-bound UvrD helicase . In excess UvrD at low DNA concentrations (1 nM), the bulk of the DNA is unwound rapidly by pre-bound UvrD complexes upon addition of ATP, but with time-courses that display a distinct lag phase for formation of fully unwound DNA, indicating that unwinding occurs in discrete steps, with a "step size" of four to five base-pairs as previously reported . Optimum unwinding by pre-bound UvrD-DNA complexes requires a 3' single-stranded (ss) DNA tail of 36-40 nt, whereas productive complexes do not form readily on DNA with 3'-tail lengths </=16 nt . A 5'-ssDNA tail is neither sufficient nor does it stimulate unwinding, even in the presence of a 3'-ssDNA tail . Nitrocellulose filter binding studies show that UvrD binding affinity also increases with increasing 3'-ssDNA tail length, showing apparent positive cooperativity for binding to DNA with a 40 nt 3'-ssDNA tail . Single turnover DNA unwinding experiments performed at higher DNA concentrations (50 nM) show a sigmoidal dependence of the extent of unwinding on the pre-incubated {UvrD}, also indicating cooperativity . These results indicate that the form of the UvrD helicase with optimal helicase activity is oligomeric with at least two sites for binding the DNA substrate, where one site contacts regions of the 3'-ssDNA tail that are distal from the single-stranded/double-stranded DNA junction .

Arch Virol, 1999, 144(9), 1701 - 12
E . coli expressed proteins as diagnostic reagents for typing of foot-and-mouth disease virus; Suryanarayana VV et al.; Truncated proteins corresponding to the C-terminal half of VP1 of four vaccine strains and two field variants of foot-and-mouth disease virus (FMDV) were expressed in E . coli . The expressed proteins were affinity purified and their type specific reactivity was confirmed by immunoprecipitation with anti-virus antibodies . Antibodies were raised against the purified proteins in guinea pigs and the type specificity of the anti peptide antibodies was confirmed by antigen capture reverse transcription polymerase chain reaction (Ag-RT/PCR) where the sera against a particular type captured the homologous virus . Antibodies were purified by immuno-affinity chromatography and tested for specificity by various serological tests . Using the purified proteins and the antibodies raised against them, tests like ELISA, Ag-RT/PCR, and latex agglutination test (LAT) were standardized . Application of the reagents in various tests was studied by screening a few field samples and by nucleotide sequencing . Specific reactivity of antibodies raised against expressed protein was seen with both vaccine virus and field samples . Thus E . coli expressed proteins and antibodies to them may form an alternative and cheap source of diagnostic reagents . The studies showed that antibodies against peptides were mono-specific and therefore may be used in LAT for rapid typing of FMDV and Ag-RT/PCR for typing ELISA negative field samples.

Biochemistry, 1999 Oct 5, 38(40), 13367 - 78
Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E . coli, a TIM barrel protein; Gualfetti PJ et al.; The urea-induced equilibrium unfolding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli can be described by a four-state model, N right harpoon over left harpoon I1 right harpoon over left harpoon I2 right harpoon over left harpoon U, involving two highly populated intermediates, I1 and I2 {Gualfetti, P . J., Bilsel, O., and Matthews, C . R . (1999) Protein Sci . 8, 1623-1635} . To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques . Size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of approximately 24 A for the native state of alphaTS . The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 A, consistent with the value predicted from the X-ray structure and the Stokes radius . As the equilibrium is shifted to favor I1 at approximately 3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from approximately 38 to approximately 52 A . Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self-association of the I1 species at the relatively high concentrations required by those techniques . Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to approximately 54 A at 8 M urea . The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical for the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC . Thermodynamic parameters extracted from the SEC data for the N right harpoon over left harpoon I1 and I1 right harpoon over left harpoon I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model . The lack of sensitivity to the I2 right harpoon over left harpoon U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable . Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea {Saab-Rincon, G., Gualfetti, P . J., and Matthews, C . R . (1996) Biochemistry 35, 1988-1994} develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations . Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form . By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respective conformational ensembles.

FEBS Lett, 1999 Oct 15, 459(3), 386 - 90
Purification and functional reconstitution of a truncated human Na(+)/glucose cotransporter (SGLT1) expressed in E . coli; Panayotova-Heiermann M et al.; A truncated human Na(+)/glucose cotransporter (C(5), residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography . The truncated transporter (C(5)) was cleaved from GST-C(5) by Factor Xa proteolysis and purified by gel filtration chromatography . Up to 1 mg of purified GST-C(5) was obtained from 1 l bacterial culture . Reconstitution of both GST-C(5) and C(5) proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of {(3)H}D-glucose into C(5)-proteoliposomes than into liposomes . Transport was stereospecific, saturable, and inhibited by phloretin . These properties are similar to those obtained for C(5) in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway.

J Mol Biol, 1999 Oct 1, 292(4), 819 - 25
Mitochondrial localization and oligomeric structure of HClpP, the human homologue of E . coli ClpP; de Sagarra MR et al.; A bacterially expressed recombinant HClpP protein, the human homologue of Escherichia coli ClpP protease, was used to obtain specific polyclonal antibodies . Those antibodies identify a 26 kDa polypeptide in mitochondrial subcellular fractions of rat and human liver . Immunofluorescence and electron microscopic studies demonstrate that the mammalian homologue of ClpP is located in the mitochondrial matrix with a tendency to be found in association with the inner mitochondrial membrane . An HClpP recombinant protein with a truncated NH2terminus (missing the first 58 amino acid residues) shows a molecular mass of 26 kDa under denaturing conditions . This N-truncated HClpP recombinant protein shows a native molecular mass of 340 kDa that is identical with the native molecular mass of the partially purified protein from rat liver mitochondria . Electron microscopy shows that the N-truncated recombinant HClpP has a ring shape with seven identical morphological units in the periphery, exhibiting a 7-fold symmetry . The native molecular mass and the electron microscopic studies suggest that mitochondrial ClpP is composed of two heptameric rings with 7-fold symmetry, similar to E . coli ClpP.

J Mol Biol, 1999 Sep 3, 291(5), 1169 - 79
Functional reconstitution and characterization of AqpZ, the E . coli water channel protein; Borgnia MJ et al.; Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities . Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system . 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of approximately 2.5 mg/l of culture . The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing . Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer . Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH . Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1% SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers . Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS . Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability (pf > or = 10 x 10(-14) cm3 s-1 subunit-1) and low Arrhenius activation energy (Ea = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1) . No permeation by glycerol, urea or sorbitol was detected . Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies.

Free Radic Res, 1999 Oct, 31(4), 357 - 65
Mouse glutaredoxin - cDNA cloning, high level expression in E . coli and its possible implication in redox regulation of the DNA binding activity in transcription factor PEBP2; Nakamura T et al.; We have isolated a cDNA encoding glutaredoxin (GRX) from a mouse splenic cDNA library . This cDNA encoded a protein of 107 amino acids with a calculated molecular weight of 11.9 kDa . The deduced amino acid sequence of glutaredoxin in mouse was highly homologous with that in other mammals (81-89%), containing a putative active sequence of -Cys-Pro-Try-Cys- . Recombinant mouse glutaredoxin expressed in E . coli showed glutathione-disulfide oxidoreductase activity with beta-hydroxyethyl disulfide as its substrate, whereas mutant glutaredoxin (Cys 22, Cys 25 to Ser) showed no activity . In electrophoretic mobility shift assay, we proved that wild type GRX, not mutant one, recovered the DNA-binding activity of a transcription factor, PEBP2, oxidized by diamide . This showed that GRX may be involved in the redox regulation of the DNA-binding activity of PEBP2 as is the case with thioredoxin.

Am J Respir Crit Care Med, 1999 Oct, 160(4), 1171 - 8
Severe microcirculatory abnormalities elicited by E . coli hemolysin in the rabbit ileum mucosa; Mayer K et al.; Decreased capillary flow and heterogeneity of microvascular perfusion are hallmarks of septic circulatory disturbances, and the gastrointestinal mucosa is considered to be particularly prone to such abnormalities . We investigated the impact of Escherichia coli hemolysin (HlyA), a medically relevant pore-forming bacterial toxin, on the mucosal microvasculature in a constant-flow blood-perfused rabbit ileum model . Microsensor techniques were employed to assess spatial distribution of mucosal hemoglobin oxygenation and relative mucosal hemoglobin content, as well as mucosal-arterial PCO(2) gap . Administration of low doses of HlyA (0.005 to 0.1 hemolytic units {HU}/ml) into the mesenteric artery provoked a transient vasoconstrictor response . Whereas physiological mucosal oxygenation is homogeneous, severe heterogeneity in capillary blood flow distribution appeared, paralleled by a marked increase in the mucosal-arterial PCO(2) gap . In addition, HlyA provoked a dose-dependent increase in relative hemoglobin concentration (rel Hb(conc)) values and edema formation, suggesting postcapillary vasoconstriction and capillary leakage . The observed changes occurred while fully maintaining mesenteric oxygen delivery . We conclude that low doses of HlyA may elicit severe mucosal microcirculatory disturbances in the rabbit ileum under maintenance of global hemodynamics, reminiscent of septic perfusion abnormalities . Pore-forming bacterial toxins may thus be considered as contributors to splanchnic mucosal damage under conditions of severe infection and sepsis.

Nat Struct Biol, 1999 Oct, 6(10), 961 - 8
Protein-nucleotide interactions in E . coli DNA topoisomerase I; Feinberg H et al.; DNA topoisomerases are the enzymes responsible for controlling and maintaining the topological states of DNA . Type IA enzymes work by transiently breaking the phosphodiester backbone of one strand to allow passage of another strand through the break . The protein has to perform complex rearrangements of the DNA, and hence it is likely that different regions of the enzyme bind DNA with different affinities . In order to identify some of the DNA binding sites in the protein, we have solved the structures of several complexes of the 67 kDa N-terminal fragment of Escherichia coli DNA topoisomerase I with mono- and trinucleotides . There are five different binding sites in the complexes, one of which is adjacent to the active site . Two other sites are in the central hole of the protein and may represent general DNA binding regions . The positions of these sites allow us to identify different DNA binding regions and to understand their possible roles in the catalytic cycle.

Nat Struct Biol, 1999 Oct, 6(10), 918 - 22
Conformational changes in E . coli DNA topoisomerase I; Feinberg H et al.; DNA topoisomerases are the enzymes responsible for maintaining the topological states of DNA . In order to change the topology of DNA, topoisomerases pass one or two DNA strands through transient single or double strand breaks in the DNA phosphodiester backbone . It has been proposed that both type IA and type II enzymes change conformation dramatically during the reaction cycle in order to accomplish these transformations . In the case of Escherichia coli DNA topoisomerase I, it has been suggested that a 30 kDa fragment moves away from the rest of the protein to create an entrance into the central hole in the protein . Structures of the 30 kDa fragment reveal that indeed this fragment can change conformation significantly . The fragment is composed of two domains, and while the domains themselves remain largely unchanged, their relative arrangement can change dramatically.

J Audiov Media Med, 1998 Dec, 21(4), 133 - 9
The role of a medical illustration department during the Lanarkshire E . coli O157 outbreak; Milligan RG et al.; In November and December 1996 an outbreak occurred of Escherichia coli (E . coli) O157 gastro-enteritis, centred on the Lanarkshire town of Wishaw, in central Scotland . The majority of resultant admissions were received by a single local general hospital . Monklands Hospital, in Airdrie, is the site of the Lanarkshire Area Infectious Diseases (ID) Unit and over the period of the outbreak one hundred and thirteen cases were admitted . This placed considerable strain on the resources of the hospital; at one stage the hospital was closed to all but emergency admissions . The hospital Medical Illustration Department was involved in a supporting role to the clinical team dealing with the outbreak as well as assisting the hospital management team with other duties . This paper gives a brief history of the event and describes the range of duties undertaken by the department during the incident . It is hoped that it will raise awareness, within the medical illustration profession, of the role that Medical Illustration Departments can play in major medical incidents.

J Membr Biol, 1999 Oct 1, 171(3), 183 - 93
Stoichiometry of the large conductance bacterial mechanosensitive channel of E . coli . A biochemical study; Sukharev SI et al.; MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress . While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M . tuberculosis reveals a pentameric structure . The present work further examines the stoichiometry of the E . coli MscL using a variety of biochemical approaches . Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels . Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product . DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker . It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts . For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species . Thus the longer spacer arm could permit both intra- and intercomplex linkages . Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel . Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers . The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization . Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric . The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor . The channel is thus a pentameric core with the 'extra' subunits left out of the functional complex . From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.

Verh K Acad Geneeskd Belg, 1999, 61(4), 489 - 515
{Genetic investigation of the resistance mechanisms of the pig against diarrhea caused by E . coli}; Peelman LJ; The aim of this study was to determine the position of the receptorgenes coding for the binding places of the K88 E . coli fimbriae, as a first step in the development of a non-invasive diagnostic test to type pigs for sensitivity for or resistance against K88 E.coli neonatal diarrhoea . Two approaches that were subsequently merged were followed . In the first one a linkage study was performed using chromosome 13 microsatellites and the with immunofluorescence determined K88ab, ac and ad phenotypes . Four of the microsatellite markers were found closely associated with the K88ab and ac locus, and these can now be used as a preliminary diagnostic tool to type for K88 resistance or sensitivity . These four markers enclose a 2 Mbp region containing the K88ab and ac receptorgenes . None of the 17 markers showed any linkage with the ad-locus indicating the ad-receptor gene is not located on chromosome 13 of the pig . The second approach was aimed at searching for genes in the chromsome 13 orthologous region of human chromosome 3 . The orthologous region could not be determined due to chromosomal rearrangements . However, two chomosomal bands, 3p26 and 3q21, could be found as converging in band 13q31 of the pig, as genes of both human regions were found in the pig in that same location . The genes in these regions can now be used to enrich the map in pig band 13q31 and to search for K88 candidate receptorgenes . Integration of the linkage map with the physical map was performed by FISH mapping some of the microsatellites and by including some of the microsatellites found near the previously FISH mapped genes in the linkage map . In conclusion, one can state that we have now in hand an excellent tool to search for the K88ab and ac receptorgenes in the 2Mbp region flanked by the four microsatellites.

Bioorg Khim, 1999 May, 25(5), 329 - 33
{Synthesis of a modified analog of a native lipotripeptide from the E . coli bacterial wall}; Usanova MP et al.; The following prospective immunomodulators, aliphatic derivatives of amino acids and peptides, were synthesized: N-palmitoyl-L-cysteine, N-palmitoyl-S-{1,2-bis(hexadecyloxycarbonyl)ethyl}-L-cysteine, N-palmitoyl-S-{1,2-bis(hexadecyloxycarbonyl)ethyl}-L-cysteinyl-L-s erine, and N-palmitoyl-S-{1,2-bis(hexadecyloxycarbonyl) ethyl}-L-cysteinyl-L-seryl-L-serine.

Biochemistry, 1999 Sep 21, 38(38), 12258 - 65
Cytidine deaminases from B . subtilis and E . coli: compensating effects of changing zinc coordination and quaternary structure; Carlow DC et al.; Cytidine deaminase from E . coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom . Cytidine deaminase from B . subtilis is a tetramer of identical subunits (M(r) = 14 800) . After purification from an overexpressing strain, the enzyme from B . subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy . Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine . A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E . coli, strongly suggests that in the enzyme from B . subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) {Song, B . H., and Neuhard, J . (1989) Mol . Gen . Genet . 216, 462-468} . This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E . coli (His-102, Cys-129, and Cys-132) . Instead, the B . subtilis and E . coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude . In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit . Sequence alignments, together with model building, suggest one possible mechanism of compensation.

Mol Cell, 1999 Aug, 4(2), 281 - 6
The dinB gene encodes a novel E . coli DNA polymerase, DNA pol IV, involved in mutagenesis; Wagner J et al.; In Escherichia coli, the dinB gene is required for the SOS-induced lambda untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed . Here, we report that the purified DinB protein is a DNA polymerase . This novel E . coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures . Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity . Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.

FEMS Microbiol Lett, 1999 Sep 1, 178(1), 13 - 8
Reconstitution of active recombinant Shiga toxin (Stx)1 from recombinant Stx1-A and Stx1-B subunits independently produced by E . coli clones; Nishikawa T et al.; Escherichia coli clones expressing recombinant Shiga toxin (Stx)1-A and recombinant Stx1-B subunits, were established . Culture supernatants of these clones were examined for inhibitory activity on in vitro protein synthesis using luciferase as a reporter enzyme . Culture supernatant of the clone expressing Stx1-A, but not Stx1-B, showed the inhibitory activity . Neither recombinant Stx1-A nor Stx1-B showed Vero cell cytotoxicity . For reconstitution of biologically active toxin, the culture supernatants of the Stx1-A clone and the Stx1-B clone were mixed . The reconstituted recombinant Stx1 showed both Vero cell cytotoxicity and inhibition of in vitro protein synthesis.

J Biotechnol, 1999 May 28, 71(1-3), 39 - 58
Characterization of stress and protein turnover from protein overexpression in fed-batch E . coli cultures; Ramirez DM et al.; A structured kinetic model that accounts for proteolytic degradation due to recombinant protein overexpression is introduced and its performance evaluated by comparison with previously reported fed-batch experimental data . This mathematical model contains an additional pool for a generic key precursor (in our case phenylalanine), an improved IPTG transport term, a phenylalanine transport term, and a variable protein turnover expression that accounts for proteolytic activity . The model predictions concerning proteolytic activity, glucose level, and cell growth are in very good agreement with an amino acid depletion hypothesis . Cultures exposed to greater stress showed higher and/or longer proteolysis, whereas less overall proteolytic activity was observed when the effect of induction was somewhat ameliorated.

Nucleosides Nucleotides, 1999 Jun-Jul, 18(6-7), 1339 - 41
Synthesis and biological evaluation of modified DNA fragments for the study of nucleotide excision repair in E . coli; Monaco V et al.; Three new cholesterol-containing phosphoramidites where synthesized and used in automated synthesis of modified DNA fragments . These cholesterol lesions are good substrates for the E . coli UvrABC endonuclease . In vitro they are incised from damaged DNA with higher efficiency in respect with the cholesterol lesions previously published.

Physiol Behav, 1999 Aug 1, 67(1), 133 - 40
Effects of social conflict on immune responses and E . coli growth within closed chambers in mice; Dreau D et al.; Social conflict has been shown to affect the neuroendocrine stress response in rodents . The current study was designed to characterize the effects of social conflict on leukocyte subset distribution and function as well as in vivo bacterial growth . Male DBA/2 mice implanted or not implanted with a closed chamber containing Escherichia coli were repeatedly challenged by temporary placement in the territory of a dominant CF-1 mouse five times a day for 2 consecutive days . Nonstressed animals were similarly handled, but were not exposed to social conflict . Effects on immune responses and E . coli growth were analyzed 13 h after the last social conflict session . Social conflict alone was associated with an increase in plasma corticosterone concentration and decreases in thymocyte numbers and splenocyte ability to proliferate in vitro in the presence of lipopolysaccharide (p < 0.05) . After social conflict, immature CD4+CD8+ thymocytes decreased, whereas mature T cells increased (p < 0.05) . In the presence of E . coli, social conflict induced a significant increase in plasma concentration of interleukin-1beta, and a decrease in the number of thymocytes and the percentage of CD4+CD8+ T cells in the thymus (p < 0.05) . In addition to the lymphocyte subpopulation changes observed with social conflict alone, the proportion of CD3+ and major histocompatibility complex (MHC) class II IAd+ cells were significantly higher in stressed mice implanted with a closed chamber containing E . coli (p < 0.05) . Social conflict tended to favor E . coli growth in the closed chamber, indicating possible direct bacterial-neuroendocrine hormone interactions . Taken together, these results suggest that stress may modulate the host immune response by altering both bacterial growth and resistance to infection.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1997 Aug, 19(4), 312 - 4
{The curative effects of anti-TNF monoclonal antibody in E . coli infected mice}; Guo X et al.; OBJECTIVE: To evaluate effects of monoclonal antibody (McAb) against TNFa on E . coli infected mice . METHODS: 30 mice (10 week old, Kunming) were divided equally into three groups . The first group, as a control, 200 microliters saline (NS)/mouse was injected intravenously; Second group (untreated group), only E . coli (10(7) organisms/200 microliters NS) were injected intra-abdominaly; Third group (treated group), E . coli (10(7) organisms) were injected intra-abdominal and McAb against TNF alpha 2 mg/kg dissolved in 200 microliters NS were injected intravenously . After 24 h, observed the survival rate, compared the serum TNF level in blood and investigated pathology of intestine, lung and liver . RESULTS: There was a higher survival rate in treated group, with the serum TNF level lowered significantly, and the untreated mice had severe pathologic changes in viscera . CONCLUSION: Using anti-TNF alpha McAb was effective in reducing mortality rate in mice after infected with E . coli, but could not prevent the pathologic changes.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1997 Feb, 19(1), 29 - 34
{cDNA cloning of human stem cell factor and its high level expression in E . coli}; Chen W et al.; The total RNA of HepG2 cell was extracted as the template by ultrocentrifuge method . The full length cDNA (0.8 kb) encoding the human stem cell factor (hSCF) was amplified by RT-PCR method . The cDNA encoding mature hSCF (0.5 kb) was sequenced and was recombined into the expression vector (PBV-220) . The expression level of rhSCF in E . coli DH5 alpha was about 15% of the total protein.

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