Escherichia coli

EMBO J, 1999 Aug 16, 18(16), 4464 - 75
Wrapping of DNA around the E.coli RNA polymerase open promoter complex; Rivetti C et al.; High-resolution atomic force microscopy (AFM) and biochemical methods were used to analyze the structure of Escherichia coli RNA polymerase.sigma(70) (RNAP) open promoter complex (RP(o)) . A detailed analysis of a large number of molecules shows that the DNA contour length of RP(o) is reduced by approximately 30 nm (approximately 90 bp) relative to the free DNA . The DNA bend angle measured with different methods varied from 55 to 88 degrees . The contour length reduction and the DNA bend angle were much less in inactive RNAP-DNA complexes . These results, together with previously published observations, strongly support the notion that during transcription initiation, the promoter DNA wraps nearly 300 degrees around the polymerase . This amount of DNA bending requires an energy of 60 kJ/mol . The structural analysis of the open promoter complexes revealed that two-thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one-third is part of the downstream region . Based on these data, a model of the sigma(70).RP(o) conformation is proposed.

Mol Cell Probes, 1999 Aug, 13(4), 291 - 302
Semi-automated fluorogenic PCR assays (TaqMan) forrapid detection of Escherichia coli O157:H7 and other shiga toxigenic E . coli; Sharma VK et al.; Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-O157:H7 Shiga toxin-producing E . coli (STEC) was achieved using fluorogenic polymerase chain reaction (PCR) . These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence genes stx1, stx2 and eaeA, respectively, using specific primers . The fluorogenic probes were used for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene of EHEC O157:H7 . For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes . In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions . The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1, stx2 and eaeA . The multiplex assay detected all STEC harbouring any combination of three virulence genes . Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC . Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively . These assays can be completed within 8-10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1 and stx2 of STEC and eaeA of EHEC O157:H7

Biochemistry, 1999 Aug 10, 38(32), 10567 - 77
Backbone dynamics of the N-terminal domain in E . coli DnaJ determined by 15N- and 13CO-relaxation measurements; Huang K et al.; The backbone dynamics of the N-terminal domain of the chaperone protein Escherichia coli DnaJ have been investigated using steady-state 1H-15N NOEs, 15N T1, T2, and T1 rho relaxation times, steady-state 13C alpha-13CO NOEs, and 13CO T1 relaxation times . Two recombinant constructs of the N-terminal domain of DnaJ have been studied . One, DnaJ(1-78), contains the most conserved "J-domain" of DnaJ, and the other, DnaJ(1-104), includes a glycine/phenylalanine rich region ("G/F" region) in addition to the "J-domain" . DnaJ(1-78) is not capable of stimulating ATP hydrolysis by DnaK, despite the fact that all currently identified sites responsible for DnaJ-DnaK interaction are located in this region . DnaJ(1-104), on the other hand, retains nearly the full ATPase stimulatory activity of full length DnaJ . Recently, a structural analysis of these two molecules was presented in an effort to elucidate the origin of their functional differences {Huang, K., Flanagan, J . M., and Prestegard, J . H . (1999) Protein Science 8, 203-214} . Herein, an analysis of dynamic properties is presented in a similar effort . A generalized model-free approach with a full treatment of the anisotropic overall rotation of the proteins is used in the analysis of measured relaxation parameters . Our results show that internal motions on pico- to nanosecond time scales in the backbone of DnaJ(1-78) are reduced on the inclusion of the "G/F" region, while conformational exchange on micro- to millisecond time scales increases . We speculate that the enhanced flexibility of residues on the slow time scale upon the inclusion of the "G/F" region could be relevant to the ATPase stimulatory activity of DnaJ if an "induced-fit" mechanism applies to DnaJ-DnaK interactions.

Gene Ther, 1999 Mar, 6(3), 442 - 7
Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E . coli DH10B using an inducible homologous recombination system; Narayanan K et al.; Gene therapy studies require techniques that allow alteration of human genomic DNA sequences . Bacterial artificial chromosome cloning systems (BACs/PACs) bridge the gap between vectors with small inserts and yeast artificial chromosomes (YACs) . We report the use of a second generation BAC vector, pEBAC, containing eukaryotic selectable markers and combining some of the best features of the BAC, PAC and HAEC systems, into which a 185 kb sequence containing the human beta-globin gene cluster was retrofitted . To permit the introduction of mutations corresponding to those causing human pathology, we have adapted an inducible homologous recombination system for use in E . coli DH10B cells, the host strain for BACs and PACs . Using this system, we have introduced PCR fragments carrying a selectable marker and a reporter gene downstream of the IVS-110 splicing mutation into a specific site within the beta-globin gene sequence . The use of this inducible system minimises the risk of unwanted rearrangements by recombination between repetitive elements and allows the introduction of relevant modifications or reporters at any specific sequence within BACs/PACs in E . coli DH10B cells.

Nucleosides Nucleotides, 1999 Apr-May, 18(4-5), 745 - 57
Gene therapy of cancer: activation of nucleoside prodrugs with E . coli purine nucleoside phosphorylase; Secrist JA 3rd et al.; During the last few years, many gene therapy strategies have been developed for various disease targets . The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal . One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir . We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes . Specifically, our approach involves the delivery of E . coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound . This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

Nucleosides Nucleotides, 1999 Apr-May, 18(4-5), 861 - 2
On the catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from E . coli; Allart B et al.; AdoHcy/MTA nucleosidase has been under scrutiny in a series of studies to explore its catalytic mechanism.

J Membr Biol, 1999 Jul 15, 170(2), 135 - 45
Gating kinetics of E . coli poly-3-hydroxybutyrate/polyphosphate channels in planar bilayer membranes; Das S et al.; Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers . Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 +/- 3 pS), and a major subconductance state (56 +/- 2 pS) . Other subconductance states and full closures are rare (<0.5% of total time) . Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure . Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer . The fraction of time spent in each conductance level was strongly voltage-sensitive . For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials . Analysis of open time distributions revealed existence of two kinetically distinct states for each level . The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive . A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed.

FEBS Lett, 1999 Jul 16, 455(1-2), 130 - 4
Characterization of the domains of E . coli initiation factor IF2 responsible for recognition of the ribosome; Moreno JM et al.; We have studied the interactions between the ribosome and the domains of Escherichia coli translation initiation factor 2, using an in vitro ribosomal binding assay with wild-type forms, N- and C-terminal truncated forms of IF2 as well as isolated structural domains . A deletion mutant of the factor consisting of the two N-terminal domains of IF2, binds to both 30S and 50S ribosomal subunits as well as to 70S ribosomes . Furthermore, a truncated form of IF2, lacking the two N-terminal domains, binds to 30S ribosomal subunits in the presence of IF1 . In addition, this N-terminal deletion mutant IF2 possess a low but significant affinity for the 70S ribosome which is increased by addition of IF1 . The isolated C-terminal domain of IF2 has no intrinsic affinity for the ribosome nor does the deletion of this domain from IF2 affect the ribosomal binding capability of IF2 . We conclude that the N-terminus of IF2 is required for optimal interaction of the factor with both 30S and 50S ribosomal subunits . A structural model for the interaction of IF2 with the ribosome is presented.

Anal Chem, 1999 Jul 15, 71(14), 2858 - 65
BIA/MS of epitope-tagged peptides directly from E . coli lysate: multiplex detection and protein identification at low-femtomole to subfemtomole levels; Nelson RW et al.; The use of biomolecular interaction analysis mass spectrometry to selectively isolate, detect, and characterize epitope-tagged peptides present in total cell lysates is demonstrated . Epitope-tagged tryptic peptides were captured via affinity interactions with either chelated Ni2+ or monoclonal antibodies and detected using surface plasmon resonance biomolecular interaction analysis (SPR-BIA) . After SPR-BIA the tagged peptides were either eluted from the biosensor chips for mass spectrometric analysis or analyzed directly from the biosensor chip using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) . Protein database searches were performed using the masses of the tagged tryptic peptides, resulting in identification of the protein into which the epitope tag was inserted . Detection limits for both SPR-BIA and MALDI-TOF were at the low-femtomole to subfemtomole level . The approach represents a (multiplexed) high-sensitivity chip-based technique capable of identifying epitope-tagged proteins as they are present in complex mixtures.

Mol Cells, 1999 Jun 30, 9(3), 338 - 43
Nucleotide sequence analysis of the 3'-terminal region of two Korean isolates of lily symptomless Carlavirus and expression of the coat protein in E . coli; Ahn HI et al.; The 3'-terminal regions of the genomic RNAs of two Korean isolates of the lily symptomless Carlavirus (LSV), LSV-Ko and LSV-KII, were cloned and their nucleotide sequences were determined . The nucleotide sequence analysis and protein analysis by the Western blot revealed that E . coli expressed a 32-kDa protein that is the viral coat protein (CP) for the LSV . The two Korean strains share 98.4% and 98.3% sequence identities at the nucleotide and amino acid levels, respectively . The CP gene of LSV-Ko showed 99.1% and 87.0% nucleotide sequence identities, and 99.0% and 96.6% amino acid sequence identities with those of the Netherlands and the Japanese LSV strains, respectively . A pairwise amino acid sequence comparison revealed a sequence similarity of 29.6% to 69.8% between LSV-Ko and other species of the carlavirus . The 16 kDa protein of LSV-Ko shares 17.6% to 42.7% amino acid similarity with those of 8 other the carlaviruses, and they are variable in the N-terminal region . The Cys repeated zinc finger nucleic acid binding domain was found in the 16 kDa protein for all of the LSV strains . Sequence comparisons of the 7 kDa protein of LSV in the strain level showed significant identities from 100.0% to 98.4% . LSV-Ko shares 21.9% to 42.2% amino acid similarity with those of 8 other carlaviruses, 4 members of the potexviruses, and a closterovirus . LSV is closely related to blueberry scorch virus (BISV) based upon the phylogenetic tree analyses of the three proteins, indicating LSV to be a quite distinct member of the genus Carlavirus.

Infect Immun, 1999 Aug, 67(8), 4260 - 3
The cloned locus of enterocyte effacement from enterohemorrhagic Escherichia coli O157:H7 is unable to confer the attaching and effacing phenotype upon E . coli K-12; Elliott SJ et al.; The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic Escherichia coli (EHEC) O157:H7 possesses the same genes in identical order and orientation as the LEE of enteropathogenic E . coli (EPEC) O127:H6 but is unable to form attaching and effacing (A/E) lesions or to secrete Esp proteins when it is cloned in an E . coli K-12 background . The A/E phenotype could not be restored by trans complementation with a variety of cloned EPEC LEE fragments, suggesting functional and/or regulatory differences between the LEE pathogenicity islands of EPEC O127:H6 and EHEC O157:H7.

Structure Fold Des, 1999 Jun 15, 7(6), 681 - 90
NMR structure of the N-terminal domain of E . coli DnaB helicase: implications for structure rearrangements in the helicase hexamer; Weigelt J et al.; BACKGROUND: DnaB is the primary replicative helicase in Escherichia coli . Native DnaB is a hexamer of identical subunits, each consisting of a larger C-terminal domain and a smaller N-terminal domain . Electron-microscopy data show hexamers with C6 or C3 symmetry, indicating large domain movements and reversible pairwise association . RESULTS: The three-dimensional structure of the N-terminal domain of E . coli DnaB was determined by nuclear magnetic resonance (NMR) spectroscopy . Structural similarity was found with the primary dimerisation domain of a topoisomerase, the gyrase A subunit from E . coli . A monomer-dimer equilibrium was observed for the isolated N-terminal domain of DnaB . A dimer model with C2 symmetry was derived from intermolecular nuclear Overhauser effects, which is consistent with all available NMR data . CONCLUSIONS: The monomer-dimer equilibrium observed for the N-terminal domain of DnaB is likely to be of functional significance for helicase activity, by participating in the switch between C6 and C3 symmetry of the helicase hexamer.

Can J Public Health, 1999 May-Jun, 90(3), 172 - 5
The relationship between E . coli indicator bacteria in well-water and gastrointestinal illnesses in rural families; Raina PS et al.; OBJECTIVES: To determine the relationship between consumption of E . coli contaminated well-water and gastrointestinal illness in rural families . METHODS: One hundred and eighty-one families with well-water as a drinking source participated in a one-year follow-up study . Water was tested for E . coli bacteria and health outcomes were monitored for house-hold members . RESULTS: E . coli in well-water was significantly associated with gastrointestinal illness in family members, however the relationship was modified by the distance from the septic tank to the well . E . coli had an odds ratio of 2.16 {95% CI 1.04, 4.42} if the septic tank was greater than 20 metres from the well and 0.46 {95% CI 0.07, 2.95} if the septic tank was within 20 metres . CONCLUSIONS: Consumption of contaminated well-water is associated with gastrointestinal illness . E . coli can be a useful marker for detecting wells that pose a potential public health problem in rural areas.

Biotechnol Bioeng, 1999 Jun 20, 63(6), 712 - 20
Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E . coli gapA gene; Gschaedler A et al.; At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect) . To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E . coli, the effect of a pulse addition of glucose, on transcription of a cloned E . coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E . coli HB101 transformed with the plasmid pBR::EcogapA . Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose . After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced . As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased . However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions . This is shown by a strong activation of promoter P2, that is recognized by the stress specific Esigma32 RNA polymerase . During this period, the total cellular RNA content was strongly diminished . Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed . The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities . For comparison, pulse-addition experiments were also performed with serine and alanine . A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production . The implication of these data for overproduction of recombinant proteins in E . coli is discussed .

Biochemistry, 1999 Jun 15, 38(24), 7737 - 46
Synthetase recognition determinants of E . coli valine transfer RNA; Horowitz J et al.; We have studied the interactions between Escherichia coli tRNAVal and valyl-tRNA synthetase (ValRS) by enzymatic footprinting with nuclease S1 and ribonuclease V1, and by analysis of the aminoacylation kinetics of mutant tRNAVal transcripts . Valyl-tRNA synthetase specifically protects the anticodon loop, the 3' side of the stacked T-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNAVal against cleavage by double- and single-strand-specific nucleases . Increased nuclease susceptibility at the ends of the anticodon- and T-stems in the tRNAVal.ValRS complex is indicative of enzyme-induced conformational changes in the tRNA . The most important synthetase recognition determinants are the middle and 3' anticodon nucleotides (A35 and C36, respectively); G20, in the variable pocket, and G45, in the tRNA central core, are minor recognition elements . The discriminator base, position 73, and the anticodon stem also are recognized by ValRS . Replacing wild-type A73 with G73 reduces the aminoacylation efficiency more than 40-fold . However, the C73 and U73 mutants remain good substrates for ValRS, suggesting that guanosine at position 73 acts as a negative determinant . The amino acid acceptor arm of tRNAVal contains no other synthetase recognition nucleotides, but regular A-type RNA helix geometry in the acceptor stem is essential {Liu, M., et al . (1997) Nucleic Acids Res . 25, 4883-4890} . In the anticodon stem, converting the U29:A41 base pair to C29:G41 reduces the aminoacylation efficiency 50-fold . This is apparently due to the rigidity of the anticodon stem caused by the presence of five consecutive C:G base pairs, since the A29:U41 mutant is readily aminoacylated . Identity switch experiments provide additional evidence for a role of the anticodon stem in synthetase recognition . The valine recognition determinants, A35, C36, A73, G20, G45, and a regular A-RNA acceptor helix are insufficient to transform E . coli tRNAPhe into an effective valine acceptor . Replacing the anticodon stem of tRNAPhe with that of tRNAVal, however, converts the tRNA into a good substrate for ValRS . These experiments confirm G45 as a minor ValRS recognition element.

Bioorg Khim, 1999 Mar, 25(3), 184 - 8
{Cloning of streptavidin gene from Streptomyces avidinii and its expression in Escherichia coli . Secretion of streptavidin by E . coli cells}; Veiko VP et al.; The streptavidin gene from Streptomyces avidinii was cloned, an expression plasmid constructed, and a highly effective strain producer of streptavidin created . It was shown that the leader peptide of streptavidin ensures the effective secretion of this protein into the periplasmic space of Escherichia coli cells . The degradation site of the leader peptide was detected . Upon treatment with the total fraction of proteases secreted by S . avidinii into the culture medium, "core" streptavidin was obtained, which retained the biotin-binding function.

Yi Chuan Xue Bao, 1999, 26(1), 87 - 91
{Expression of chimeric single-chain antibody with specificity for HBsAg in E . coli}; Pan H et al.; The Vk gene of MAb with specificity for HBsAg and human Ck gene have been combined to form chimeric light chain (Vk-Ck) by recombinant PCR, which has combined with VH to construct chimeric single-chain antibody(ScFv-Ck) by a linker encoding a flexible peptide{(Gly3 Ser)3} . The ScFv-Ck has been expressed in the heat-induced expression system and secreted expression system of E . coli respectively . Analysis by Western-blot and indirect ELISA shows that the ScFv-Ck product in two systems both have HBsAg-binding ability, and ScFv-Ck has been secreted from E . coli in the direction of secretion peptide.

Chem Biol, 1999 Jun, 6(6), 385 - 400
Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E . coli EntF catalyzes both elongation and cyclolactonization; Shaw-Reid CA et al.; BACKGROUND: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N-acyl-serine trilactone siderophore enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE) . To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain . RESULTS: The Ser1138-->Cys mutant (kcat decreased 1000-fold compared with wild-type EntF) releases both enterobactin (75%) and linear (DHB-Ser)2 dimer (25%) as products . The His 1271-->Ala mutant (kcat decreased 10,000-fold compared with wild-type EntF) releases only enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser)2-O-TE acyl enzyme intermediates . Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates . CONCLUSIONS: These results establish that the TE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.

Biotechnol Prog, 1999 May-Jun, 15(3), 467 - 71
Functional expression of horseradish peroxidase in E . coli by directed evolution; Lin Z et al.; In an effort to develop a bacterial expression system for horseradish peroxidase (HRP), we inserted the gene encoding HRP into the pET-22b(+) vector (Novagen) as a fusion to the signal peptide PelB . A similar construct for cytochrome c peroxidase (CcP) leads to high CcP activity in the supernatant . Expression of the wild-type HRP gene in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) yielded no detectable activity against ABTS (azinobis(ethylbenzthiazoline sulfonate)) . However, weak peroxidase activity was detected in the supernatant in the absence of IPTG . The HRP gene was subjected to directed evolution: random mutagenesis and gene recombination followed by screening in a 96-well microplate format . From 12 000 clones screened in the first generation, one was found that showed 14-fold higher HRP activity than wild-type, amounting to approximately 110 microg of HRP/L, which is similar to that reported from laborious in vitro refolding . No further improvement was obtained in subsequent generations of directed evolution . This level of expression has nonetheless enabled us to carry out further directed evolution to render the enzyme more thermostable and more resistant toward inactivation by H2O2 . These results show that directed evolution can identify mutations that assist proteins to fold more efficiently in Escherichia coli . This approach will greatly facilitate efforts to "fine-tune" those many enzymes that are promising industrial biocatalysts, but for which suitable bacterial or yeast expression systems are currently lacking.

J Vet Med Sci, 1999 Apr, 61(4), 433 - 8
Histological characteristics of canine deciduoma induced by intrauterine inoculation of E . coli suspension; Nomura K et al.; Canine deciduoma could be induced in the diestrous uterus by an intrauterine inoculation of a culture suspension of E . coli originally isolated from naturally occurring canine pyometra . These deciduomas had the same histological findings as those of naturally occurring canine pyometra with so called "Swiss cheese endometrium" . This suggests a possibility that the canine pyometra is a kind of naturally occurring decidual reaction (deciduoma) induced by one of several triggers such as bacterial infection.

J Mol Biol, 1999 May 28, 289(1), 57 - 67
Biophysical analysis of the interaction of human ifnar2 expressed in E . coli with IFNalpha2; Piehler J et al.; Type I interferons are cytokines which activate an anti-viral response by binding to two specific cell surface receptors, ifnar1 and ifnar2 . Here, we report purification and refolding of the extracellular part of human ifnar2 (ifnar2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha2 (IFNalpha2) . The 25 kDa, non-glycosylated ifnar2-EC is a stable, fully active protein, which inhibits antiviral activity of IFNalpha2 . The stoichiometry of binding IFNalpha2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection . The affinity of this interaction is 10 nM, which is similar to the affinity measured for the cell surface-bound ifnar2 receptor . No difference in affinity was found throughout various assays using optical detection as BIAcore or reflectometric interference spectorscopy . However, the binding kinetics as measured in homogeneous phase by fluorescence de-quenching was about three times faster than that measured on a sensor surface . The rate of complex formation is relatively high compared to other cytokine-receptor interactions . The salt dependence of the association kinetics suggest a limited but significant contribution of electrostatic forces towards the rate of complex formation . The dissociation constant increases with decreasing pH according to the protonation of a base with a pKa of 6.7 . The surface properties of the IFNalpha2 binding surface on ifnar2 were interpreted according to the pH and salt dependence of the interaction .

Aviakosm Ekolog Med, 1999, 33(1), 61 - 2
{Effect of electrostatic field on bacteria E . coli}; Umarov GR et al.; On the basis of the quantum mechanical theory of phase transitions and chemical reactions developed by the authors it is shown that the mentioned processes are controlled by the definite active centers . The electrostatic fields (without electric current included) are capable of changing the degree of activity and structure of these centers and so are capable of controlling the results of processes under study . The reported experimental studies performed on the living objects validate this hypothesis . It was concluded that electrostatic fields (natural and technogenic) exert some action on life activity of the organisms and therefore it is necessary to provide the proper optimal conditions for the life activity in the inhabited spacecraft . For this purpose the special equipment is proposed by the authors.

J Mol Biol, 1999 Apr 30, 288(2), 255 - 74
A two-site kinetic mechanism for ATP binding and hydrolysis by E . coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide; Hsieh J et al.; Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis . We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA . Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol) . Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically . Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1) . However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP . Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses . The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence . The second Trp fluorescence quenching phase is associated with binding of a second ATP . The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi) . Binding of the second ATP then leads to the steady-state ATP cycle . As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding . We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism .

Gene Ther, 1998 Aug, 5(8), 1061 - 9
Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E . coli nitroreductase and CB1954; McNeish IA et al.; Expression of the E . coli enzyme nitroreductase (NTR) in tumour cells enables them to activate the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide), leading to interstrand DNA crosslinking and cell death . Using transfected or retrovirally transduced SKOV3 ovarian carcinoma cell clones, we show a strong correlation between sensitivity to CB1954 and level of NTR enzyme activity . Importantly for clinical application in ovarian cancer, a cisplatin-resistant ovarian tumour cell line remains as susceptible to the NTR-dependent cytotoxicity of CB1954 as parental cells . In mixed populations of NTR-expressing and non-expressing cells, we observe a marked 'bystander killing' effect with this system . The use of NTR-encoding retroviruses from clonal producer cell lines at titres of 5 x 10(5) c.f.u./ml to transduce either established or low passage primary ovarian carcinoma lines only achieves an average 10-fold sensitisation of the cultures at gene transfer efficiencies of 15-25% . Concentration of the retrovirus to 3 x 10(7) c.f.u./ml elevates gene transfer to 80-90% in a single exposure to target cells, resulting in up to 500-fold sensitisation of the entire, unselected SKOV3 population to CB1954 . In an initial investigation of NTR/CB1954 for the treatment of tumours in vivo, we observe regression of tumours expressing NTR following administration of CB1954, resulting in significantly increased median survival.

Vet Microbiol, 1999 Apr 1, 66(2), 125 - 34
Identification of a new Escherichia coli She haemolysin homolog in avian E . coli; Reingold J et al.; Haemolysin is one type of virulence factor that assists in the pathogenesis of Escherichia coli . Currently, hemolytic activity in E . coli has been attributed to haemolysin genes found in either uropathogenic or enterohemorrhagic E . coli . Both haemolysins are classified as RTX toxins because they both have repeats in toxin domains and share similar operon organization, sequence homology, and mechanisms of action . Haemolytic avian E . coli isolates, however, lack either E . coli haemolysin gene . To investigate the avian E . coli haemolysin, a genomic library was made from an avian pathogenic E . coli . A haemolytic clone that was isolated was shown to contain homology with sheA, an E . coli K- 12 gene which causes haemolysis when present in high copy number . The cloned haemolysin gene, hlyE, lacked the conserved amino acid sequence and accessory genes common to all RTX toxins . DNA hybridizations and polymerase chain reaction amplifications showed that the nucleotide sequences homologous to hlyE were not present in a collection of three O157: H7 E . coli, five haemolytic canine uropathogenic E . coli, one haemolytic O26 E . coli, and three haemolytic avian pathogenic E . coli . Thus we have identified a new E . coli haemolysin distinct from the RTX haemolysins and have shown that some avian pathogenic E . coli possess a haemolysin with no apparent homology to hlyE or RTX haemolysins.

Artif Cells Blood Substit Immobil Biotechnol, 1999 May, 27(3), 291 - 301
Growth kinetics of genetically engineered E . coli DH 5 cells in artificial cell APA membrane microcapsules: preliminary report; Prakash S et al.; This paper describes the growth kinetics of genetically engineered E . coli DH5 cells inside the APA membrane artificial cells . The APA microcapsule membrane found does not significantly affects the growth of the encapsulated E . coli DH5 cells . The total protein production of the E . coli DH5 cells inside the APA microcapsules were not significantly different from that of the bacterial cells grown in the free bacterial media . The result also show that the log phase APA artificial cells containing genetically engineered E . coli DH5 would be highly effective for the conversion of various external metabolites.

J Biomol Struct Dyn, 1999 Feb, 16(4), 825 - 31
DNA bendability--a novel feature in E . coli promoter recognition; Ozoline ON et al.; The distribution of deformable base-pair steps in the structure of bacterial promoters is analyzed with respect to their possible structural and functional role . A regular positioning of TA and TG stacks is detected with the best fit period 5.6 bp . This value is interpreted as a half of the sequence period 11.2 bp, somewhat higher than the structural helical repeat of B-DNA (10.55 bp) . The difference, +0.65 bp, suggests a sequence-dependent helical writhe of the promoter DNA--a right-handed superhelix . Apparently, to favour rotational setting of DNA on the surface of RNA polymerase the flexible steps deformable largely towards the grooves, follow the half-period spacing . Such rotational setting is consistent with the DNase I footprinting data . Periodical distribution of deformable base-pair stacks shows negative correlation with the presence of -35 canonical hexamer, suggesting the functional significance of this novel element for promoter recognition . The RNA polymerase--DNA recognition is discussed as interaction of distributional type that involves many elements of different nature which are in partially compensatory relations.

Biochimie, 1999 Jan-Feb, 81(1-2), 15 - 25
Nucleotide excision repair: from E . coli to man; Petit C et al.; Nucleotide excision repair is both a 'wide spectrum' DNA repair pathway and the sole system for repairing bulky damages such as UV lesions or benzo{a}pyrene adducts . The mechanisms of nucleotide excision repair are known in considerable detail in Escherichia coli . Similarly, in the past 5 years important advances have been made towards understanding the biochemical mechanisms of excision repair in humans . The overall strategy of the repair is the same in the two species: damage recognition through a multistep mechanism involving a molecular matchmaker and an ATP-dependent unwinding of the damaged duplex; dual incisions at both sides of the lesion by two different nucleases, the 3' incision being followed by the 5'; removal of the damaged oligomer; resynthesis of the repair patch, whose length matches the gap size . Despite these similarities, the two systems are biochemically different and do not even share structural homology . E . coli excinuclease employs three proteins in contrast to 16/17 polypeptides in man; the excised fragment is longer in man: the procaryotic excinuclease is not able by itself to remove the excised oligomer whereas the human enzyme does . Thus, the excinuclease mode of action is well conserved throughout evolution, but not the biochemical tools: this represents a case of evolutionary convergence.

Protein Sci, 1999 Jan, 8(1), 203 - 14
The influence of C-terminal extension on the structure of the "J-domain" in E . coli DnaJ; Huang K et al.; Two different recombinant constructs of the N-terminal domain in Escherichia coli DnaJ were uniformly labeled with nitrogen-15 and carbon-13 . One, DnaJ(1-78), contains the complete "J-domain," and the other, DnaJ(1-104), contains both the "J-domain" and a conserved "G/F" extension at the C-terminus . The three-dimensional structures of these proteins have been determined by heteronuclear NMR experiments . In both proteins the "J-domain" adopts a compact structure consisting of a helix-turn-helix-loop-helix-turn-helix motif . In contrast, the "G/F" region in DnaJ(1-104) does not fold into a well-defined structure . Nevertheless, the "G/F" region has been found to have an effect on the packing of the helices in the "J-domain" in DnaJ(1-104) . Particularly, the interhelical angles between Helix IV and other helices are significantly different in the two structures . In addition, there are some local conformational changes in the loop region connecting the two central helices . These structural differences in the "J-domain" in the presence of the "G/F" region may be related to the observation that DnaJ (1-78) is incapable of stimulating the ATPase activity of the molecular chaperone protein DnaK despite evidence that sites mediating the binding of DnaJ to DnaK are located in the 1-78 segment.

Vopr Med Khim, 1999 Jan-Feb, 45(1), 24 - 9
{Isolation and purification of recombinant truncated (delta2-27) cytochrome P450 2B4 expressed in E . coli cells as a fusion protein with glutathione-S-transferase}; Sokolov NN et al.; This paper describes the modification of the method by Coon and Pernecky (Meth . Enzymol . 1996, 272, 25-34) for purification of truncated (delta 2-27) recombinant form of cytochrome P450 2B4 expressed in E . coli as fusion protein with glutathione-S-transferase . The modifications included optimisation of conditions for proteolytic reaction of fusion protein with thrombine, removal of this protease from purified cytochrome P450 preparations using column chromatography on hydroxyapatite, introduction of the additional step for obtaining of spheroplasts using of lysozyme, and optimisation of conditions for enzyme stabilisation during of its purification and storage . The overall yield of purified cytochrome was 20% and the specific content of P450 was 14,5 nmol/mg protein was measured . This method is suitable for large-scale isolation of high purified cytochromes P450 which are necessary for study of structure-functional relationships of this hemoprotein with protein partners as well as for investigation of its structure and mechanism of action.

Biochem Mol Biol Int, 1999 Feb, 47(2), 217 - 25
Cloning, sequence analysis and expression in E . coli of the cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas; Fan CY et al.; The cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas was cloned and sequenced . The length of the cDNA is 923bp which includes 120bp of noncoding region and 780bp of coding region . Pallabin was synthesized as a prozymogen with 260 amino acids, which includes a signal peptide of 18 amino acids, a proposed propeptide of 6 amino acids and a matured peptide of 236 amino acids . Pallabin exhibits a strong amino acid similarity to the serine proteases isolated from other snake venoms . It contains 12 cysteins which form 6 disulfide bridges . Like other serine proteases, it also has three conserved catalytically active sites: His41, Asp86 and Ser182 . To our knowledge, this study is the first report concerning the cDNA of a thrombin-like enzyme from Agkistrodon halys pallas . The cDNA was cloned into the expression plasmid pT7ZZa and expressed in E.coli . The recombinant pallabin immunologically reacted with its specific antibody.

J Biotechnol, 1999 Feb 19, 68(2-3), 101 - 13
Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E . coli; Wilms B et al.; An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E . coli and the nucleotide sequence was determined . After expression of the gene in E . coli the enzyme was purified to homogeneity and characterized . The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g . N-carbamoyl-tryptophan . Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates . The N-carbamoylase of A . aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected . The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3 . The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively . At 37 degrees C the enzyme was completely stable for several days.

Biotechnol Bioeng, 1999 May 20, 63(4), 392 - 400
Expression of soluble and catalytically active plant (monocot) beta-glucosidases in E . coli; Cicek M et al.; Complementary DNAs encoding mature beta-glucosidase proteins Glu1 and Glu2 of maize were amplified by the polymerase chain reaction (PCR) and cloned into the expression vector pET21a . Both Glu1 and Glu2 isozymes were expressed in high yield ( approximately 3.8% of the total soluble protein and 32% of the total expressed protein) in E . coli . Recombinant enzymes were active on a variety of artificial and natural substrates at levels similar to those of their native counterparts isolated from maize seedlings . Western blot analysis confirmed that both recombinant isozymes were immunoreactive with maize anti-beta-glucosidase sera and their molecular sizes were identical to those of the native maize Glu1 and Glu2 isozymes . Zymogram assays in native gels revealed that recombinant enzymes had the same electrophoretic mobility and substrate specificity as their native counterparts .

Biotechnol Bioeng, 1998 Jun 5, 58(5), 536 - 40
Optimized release of recombinant proteins by ultrasonication of E . coli cells; Feliu JX et al.; The release kinetics of beta-galactosidase protein have been determined during small-scale ultrasonication of E . coli cells . Among several studied parameters, ionic strength and cell concentration have the least influence on the rate of protein recovery, whereas sample volume and acoustic power dramatically affect the final yield of soluble protein in the cell-free fraction . The analysis of these critical parameters has prompted us to propose a simple model for E . coli disintegration that only involves acoustic power and sample volume, and which allows prediction of optimal sonication times to recover significant amounts of both natural and recombinant proteins in a given set of relevant conditions .

Biotechnol Bioeng, 1998 May 20, 58(4), 356 - 65
Biotransformation of octane by E . coli HB101{pGEc47} on defined medium: octanoate production and product inhibition; Rothen SA et al.; E . coli HB101{pGEc47}, which is able to convert octane to octanoate, but cannot oxidize octanoate further, was grown on defined medium with glucose as carbon source in batch and continuous culture . The biomass yield on glucose decreased from 0.32 +/- 0.02 g g-1 in aqueous cultivations to 0.25 +/- 0.02 g g-1 in the presence of octane . Maximal octanoate productivities of 0.6 g L-1 h-1 were the same as found in cultivations on complex medium . The glucose-based carbon recovery in these experiments was 99 +/- 4% (in extreme, between 90% and 105%) . An increase of the octane feed from 1% to 2% (v/v) or more led to washout of cells . This effect was reversible when the octane feed was decreased to its initial value of 1% . Analysis of experimental data by model simulation strongly suggested that washout was due to inhibition by octanoate only . Pulses of octanoate to a continuous culture grown on aqueous media were applied to analyze the inhibition further . Inhibition by acetate was not significant, but its presence in the medium reflected a physiological state that made the cells more sensitive to octanoate inhibition . Model simulation with linear inhibition kinetics could perfectly predict glucose consumption and the resulting glucose concentration . The linear type of inhibition was confirmed by a variety of batch experiments in the presence of different concentrations of octanoate . The glucose-based specific growth rate, mu, decreased linearly with increasing concentrations of octanoate and became zero at a threshold concentration pmax of 5.25 +/- 0.25 g L-1 .

Front Biosci, 1999 Apr 01, 4, D394 - 407
Molecular surface sequence analysis of several E . coli enzymes and implications for existence of casein kinase-2 bacterial predecessor; Torshin I; Casein kinase-2 (CK2) is known as pleiotropic eukaryotic protein kinase that phosphorylates significant number of cellular proteins . Not all functions of the protein were registered up to the present time . However, it is known that this Ser/Thr-specific kinase is involved in the cell cycle progression and is essentially required for the eukaryotic cell viability . Fully automated molecular surface analysis procedure for identification of functionally significant surface residues and sequences on the base of protein spatial structure was elaborated . Using the elaborated procedure, several E . coli enzymes spatial structures and sequences were investigated . It was found that most of the casein kinase 2 potential sites found in sequences of enzymes are accessible for modification . Four of the 5 structures studied have CK2 consensus sites that may definitely influence the activity of the enzyme upon phosphorylation . Some of the potential "CK2-sites" has amino acid contents characteristic for physiological substrates of casein kinase 2 in eukaryotes . The main point of the elaborated method and the structural evidence for existence of a putative casein kinase E . coli predecessor or a protein with similar kinase activity are discussed . Physiological, biochemical, structural and evolutionary aspects of the existence of the putative predecessor are considered.

Res Microbiol, 1999 Jan-Feb, 150(1), 33 - 44
Predictive modelling of fluorescent AFLP: a new approach to the molecular epidemiology of E . coli; Arnold C et al.; Amplified fragment length polymorphism (AFLP) permits simultaneous sampling of multiple loci distributed throughout a genome, using restriction site/adaptor-specific primers under stringent conditions . Fluorescent detection instrumentation further refines this methodology, permitting internal size standards and accurate, reproducible sizing of amplified fragments . We have evaluated the potential of fluorescent AFLP (FAFLP) as a potentially definitive genotyping method for bacteria, by comparing MseI/EcoRI fragments derived experimentally from the Escherichia coli K12 MG1655 genome with those predicted by analysis of its published sequence . In silico, MseI/EcoRI digestion of this sequence produced 1200 fragments from 36 and 2151 base pairs (bp) in size . Fragment subsets which would be amplified by seven different selective (1-2 bases added to the 3' end of the core primer sequence) primer combinations were modelled . Depending on the primer pair, three to 54 fragments (range 70-400 bp) were predicted, while all seven primer pair combinations together generated 121 predicted fragments . When genomic DNA of strain MG1655 was subjected to experimental FAFLP with these seven primers, 111 correctly sized fragments were observed (+/- 1 bp) out of the 121 predicted (92% accuracy) . Twenty-five unpredicted fragments were obtained; an average of four per primer pair . The size and number of fragments in FAFLP, and their gel distribution, were dictated by the choice of restriction endonucleases and the degree of primer selectivity . Our data show that FAFLP is accurate, discriminatory, reproducible and capable of standardisation . Under agreed conditions, this method shows considerable promise as a generally applicable standardised bacterial genotyping method . The fragments predicted in silico to result from amplification of MseI/EcoRI-digested DNA with the seven primer pairs described are here used to define a prototypic FAFLP analysis of E . coli.

Biotechniques, 1999 Mar, 26(3), 518 - 22, 524, 526
Fast and accurate method for quantitating E . coli host-cell DNA contamination in plasmid DNA preparations; Smith GJ 3rd et al.; Plasmid DNA is being used successfully as a gene delivery vector in a variety of clinical applications . Similar to other pharmaceutical products for clinical use, the plasmid vectors must meet rigorous purity standards . One important contaminant is the DNA of the host cell used to produce the plasmids . We have developed a new method to accurately quantitate E . coli host-cell DNA in plasmid preparations . This method is based on kinetic PCR using the ABI PRISM 7700 with 23S rDNA as a target . This precise assay is significantly faster and has a lower limit of quantitation than the currently used Southern-based methods.

J Virol Methods, 1999 Feb, 77(2), 189 - 97
Purification of E . coli-expressed HIS-tagged hepatitis B core antigen by Ni2+ -chelate affinity chromatography; Wizemann H et al.; Hepatitis B virus is a major cause of human liver disease . In the case of chronic infection the virus can lead to liver cancer and cirrhosis . The virion consists of an outer envelope containing lipids of the endoplasmic reticulum and virally-encoded surface proteins . This lipoprotein shell encloses the nucleocapsid or core antigen (HBcAg), which contains the viral genome . The capsid consists of dimers of a 183-residue protein, which can be divided into an assembly (residues 1-149) and a protamin-like domain (residues 150-183), responsible for polymerization into particles and RNA packaging, respectively . Upon expression of the core gene in bacteria the products are assembled into capsids resembling those of wild type particles . A purification protocol was developed for unpolymerised (dimeric) and polymerized HBcAg by fusion of six histidine residues to a C-terminal deletion mutant of the core protein allowing the isolation of the respective antigens after denaturing Ni2+-chelate affinity chromatography and renaturing dialysis . The possible incorporation of E . coli proteins during the assembly process and the inclusion of nucleic acids can be avoided . The method might be an attractive alternative to common purification protocols of hybrid virus-like particles (VLPs) for vaccine use.

Biofizika, 1998 Nov-Dec, 43(6), 1032 - 6
{Inhibition of growth of E . coli cells by anolites of sodium and potassium chloride after processing solutions in a diaphragmatic electrolyzer}; Miroshnikov AI; The relationship between the inhibitory effect of sodium chloride and potassium anolites, obtained in a diaphragm electrolyser, and the physicochemical parameters of solutions was compared with that between the inhibitory effect and physicochemical properties of hypochlorites obtained after treating the solutions in an electrolyser having no diaphragm was compared . The biological activity of solutions containing molecular chlorine, hypochlorous acid, and hypochlorite ions was determined by their effect on the growth of E . coli cells . After a 5-min incubation of cells with each of the oxidizers, the bacterial growth stopped and was not restored during one day . The conclusion is made that the oxidizers irreversibly disturb the barrier properties of cell membranes and, in some cases, destroy cells . In model solutions, as well as in solutions treated after heating on a water bath or after the addition of sodium thiosulfate, a delay in the start of E . coli growth occurs . After the lag-phase, the repair of cells sets on, and after a day the optical density of cells increases and approaches the control.

Vaccine, 1999 Jan, 17(1), 95 - 8
A model to study the effects of a viral inactivator (beta-propiolactone) on DNA ligation and gene expression in E . coli and Cos cells; Fathallah DM et al.; An experimental model to study the effects of viral inactivators on the biological properties of DNA was developed . Beta-propiolactone (betaPL) was used in this model and its effects on ligation, transfer and gene expression of naked DNA were assessed . Evidence that betaPL impairs these two major DNA functions are presented . The amounts of betaPL that alter or abolish gene expression and prevent DNA cohesive ends ligation were determined . Based on these observations, it was concluded that this experimental approach could be used to study the effects on the biological properties of DNA of other inactivators used in vaccine preparations.

Mol Cell, 1999 Feb, 3(2), 229 - 38
Core RNA polymerase from E . coli induces a major change in the domain arrangement of the sigma 70 subunit; Callaci S et al.; Luminescence resonance energy transfer measurements were used to show that binding of E . coli core RNA polymerase induced major changes in interdomain distances in the sigma 70 subunit . The simplest model describing core-induced changes in sigma 70 involves a movement of the conserved region 1 by approximately 20 A and the conserved region 4.2 by approximately 15 A with respect to conserved region 2 . The core-induced movement of region 1 (autoinhibition domain) and region 4.2 (DNA-binding domain) provides structural rationale for allosteric regulation of sigma 70 DNA binding properties by the core and suggests that this regulation may not only involve directly the autoinhibition domain of sigma 70 but also could involve a modulation of spacing between DNA-binding domains of sigma 70 induced by binding of core RNAP.

Vaccine, 1999 Feb 5, 17(5), 441 - 7
Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E . coli heat-labile enterotoxin B subunit; Bagdasarian MM et al.; Peptides KDYGASTGSSL (Epil) . SLLRRRRNGEDVSV (Epi3) and DDEIYGNDGHP (Epi6), predicted to constitute immunogenic epitopes of the hemolysin-cytotoxin ApxIA of Actinobacillus pleuropneumoniae were inserted into a surface-exposed loop of the B subunit of the E . coli heat-labile enterotoxin (EtxB) . The resulting chimeric proteins were recognized by monospecific antibodies against purified native ApxI and by convalescent sera of pigs that were positive for A . pleuropneumoniae serotype 1 . Mice anti-sera against chimeric proteins EtxB::ApxIAEpi3 and EtxB::ApxIAEpi6 reacted with purified ApxI . These results indicate that Epi3 and Epi6 regions constitute linear epitopes of the structural ApxIA protein toxin . Epitope Epi6 which is located in the structure of the glycine rich repeats in ApxI elicits the formation of hemolysin neutralizing antibodies when introduced into mice in the form of a chimeric EtxB fusion protein . We suggest that fusion of peptide sequences to EtxB is a useful tool for the analysis of epitopes of complex proteins such as RTX toxins.

J Gen Virol, 1999 Feb, 80 ( Pt 2), 291 - 6
Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E . coli; Liu BL et al.; Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprotein . Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3Cpro) sequence together with flanking sequences from a region of the viral genome 3'-distal to the putative helicase-encoding region . Expression from these vectors in E . coli resulted in discrete protein products with smaller than expected molecular sizes . This confirmed that an active viral protease was produced in E . coli and that the protease was capable of cleaving the expressed protein at defined sites . Expressed cleavage products surrounding the protease region of the viral polyprotein were separated by SDS-PAGE, transferred to PVDF membranes and subjected to N-terminal sequence analysis . Cleavage occurred at an EG dipeptide at the N terminus of the putative VPg (961E/GKNKG) and also at the protease/polymerase boundary (1280E/GGDKG) . The N terminus of the protease was released from the VPg C terminus at an EA dipeptide in the sequence 1099E/APPTL . These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products.

Gene, 1999 Mar 4, 228(1-2), 253 - 60
Identification of an attenuating region in the bovine follicle-stimulating hormone beta subunit mRNA that decreases its expression in E . coli; Samaddar M et al.; Follicle-stimulating hormone (FSH) is one of the key regulators of gonadal function in mammals . Recombinant DNA expression of this hormone has proved to be a difficult task as expression levels are invariably low, irrespective of the expression system employed . In the present study, we have attempted to identify reasons for this low expression using bacterial expression vectors, and we report here the identification of a theoretically predicted hairpin structure in the mRNA corresponding to the N-terminal portion of the mature coding portion of bFSHbeta cDNA that is responsible for attenuating its expression in E . coli . When full-length FSHbeta was expressed using the bacterial expression vector, a very low expression was obtained . However, when fragments of FSHbeta with N-terminal deletions (amino acids 24-110 and 13-110) were expressed using the same expression strategy, a 30- to 40-fold higher expression was observed . This low expression of FSHbeta could be attributed to a hairpin structure present in the first 12 codons of mature FSHbeta mRNA . Disruption of this structure without changing the amino acid sequence resulted in a higher level of expression of FSHbeta . The predicted hairpin structure, though away from the transcriptional and translational start site, was able to downregulate the expression of FSHbeta probably by impeding the movement of ribosomes.

Gene, 1999 Mar 4, 228(1-2), 23 - 31
A phagemid vector using the E . coli phage shock promoter facilitates phage display of toxic proteins; Beekwilder J et al.; Phage display is a powerful tool with which to adapt the specificity of protease inhibitors . To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector . Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library . Instead, only mutants carrying deletions or amber stop codons were found . Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed . To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed . The first vector has a lower plasmid copy number, as compared to the canonical vector . Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation . A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression . The psp promoter is induced upon helper phage infection . A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host . Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display . The inhibitor library was introduced in both new vectors . Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions . This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E . coli.

Mol Gen Genet, 1999 Feb, 261(1), 122 - 32
Antibody analysis of the localisation, expression and stability of HlyD, the MFP component of the E . coli haemolysin translocator; Pimenta AL et al.; HlyD has a single transmembrane domain (residues 59-80) and a large periplasmic domain, and is essential for the secretion of haemolysin from Escherichia coli . Using an antibody raised against HlyD, the protein was localised to the cell envelope by immunofluorescence and to the cytoplasmic membrane by sucrose gradient analysis . We have examined the stability of this protein in the presence and absence of other putative components of the translocator, HlyB and TolC . HlyD is normally highly stable but in the absence of TolC, the steady-state level of HlyD is greatly reduced and the protein has a half-life at 37 degrees C of 36 min . In the absence of HlyB, HlyD is also unstable and specific degradation products are detected, which co-fractionate with the inner membrane, indicating in this case limited cleavage at specific sites . However, the effect of removing both HlyB and TolC is not additive . On the contrary, in the absence of both HlyB and TolC the half-life of HlyD is approximately 110 min . This result shows that in the presence of HlyB removal of TolC renders HlyD more unstable than it is in the absence of both HlyB and TolC . This suggests that the presence of HlyB induces a structural change in HlyD . In addition, HlyB itself appears to be less stable in the absence of HlyD . These results are consistent with an interaction between HlyD/TolC and HlyB/HlyD . A derivative of HlyD, HlyD22, lacking the 40 N-terminal residues of HlyD assembles into the inner membrane displaying the same stability with and without HlyB as wild type HlyD does . This N-terminal region therefore appears to play no role in stable localisation but is involved in secretion, since HlyD22 is completely secretion defective . Modification of the C-terminus on the other hand completely destabilised the molecule and HlyD was not detectable in the envelope . Secretion of active haemolysin is limited to a brief period during mid to late exponential phase . In contrast, HlyD is apparently synthesised constitutively throughout the growth phase, demonstrating that the production of this component of the translocator is not the limiting factor for growth phase-dependent secretion.

Am J Physiol, 1999 Mar, 276(3 Pt 1), L540 - 7
Surfactant protein A enhances the binding and deacylation of E . coli LPS by alveolar macrophages; Stamme C et al.; Surfactant protein (SP) A and SP-D are involved in multiple immunomodulatory functions of innate host defense partly via their interaction with alveolar macrophages (AMs) . In addition, both SP-A and SP-D bind to bacterial lipopolysaccharide (LPS) . To investigate the functional significance of this interaction, we first tested the ability of SP-A and SP-D to enhance the binding of tritium-labeled Escherichia coli LPS to AMs . In contrast to SP-D, SP-A enhanced the binding of LPS by AMs in a time-, temperature-, and concentration-dependent manner . Coincubation with surfactant-like lipids did not affect the SP-A-mediated enhancement of LPS binding . At SP-A-to-LPS molar ratios of 1:2-1:3, the LPS binding by AMs reached 270% of control values . Second, we investigated the role of SP-A in regulating the degradation of LPS by AMs . In the presence of SP-A, deacylation of LPS by AMs increased by approximately 2.3-fold . Pretreatment of AMs with phosphatidylinositol-specific phospholipase C had no effect on the SP-A-enhanced LPS binding but did reduce the amount of serum-enhanced LPS binding by 50%, suggesting that a cell surface molecule distinct from CD14 mediates the effect of SP-A . Together the results for the first time provide direct evidence that SP-A enhances LPS binding and degradation by AMs.

Am J Physiol, 1999 Mar, 276(3 Pt 1), G781 - 8
Enteropathogenic E . coli attenuates secretagogue-induced net intestinal ion transport but not Cl- secretion; Hecht G et al.; Enteric bacterial pathogens often increase intestinal Cl- secretion . Enteropathogenic Escherichia coli (EPEC) does not stimulate active ion secretion . In fact, EPEC infection decreases net ion transport in response to classic secretagogues . This has been presumed to reflect diminished Cl- secretion . The aim of this study was to investigate the influence of EPEC infection on specific intestinal epithelial ion transport processes . T84 cell monolayers infected with EPEC were used for these studies . EPEC infection significantly decreased short-circuit current (Isc) in response to carbachol and forskolin, yet 125I efflux studies revealed no difference in Cl- channel activity . There was also no alteration in basolateral K+ channel or Na+-K+-2Cl- cotransport activity . Furthermore, net 36Cl- flux was not decreased by EPEC . No alterations in either K+ or Na+ transport could be demonstrated . Instead, removal of basolateral bicarbonate from uninfected monolayers yielded an Isc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished Isc . These studies suggest that the reduction in stimulated Isc is not secondary to diminished Cl- secretion . Alternatively, bicarbonate-dependent transport processes appear to be perturbed.

FEBS Lett, 1999 Feb 12, 444(2-3), 189 - 94
Disruption of substrate binding site in E . coli RNA polymerase by lethal alanine substitutions in carboxy terminal domain of the beta subunit; Polyakov A et al.; Alanine substitution of four amino acids in two evolutionarily conserved motifs, PSRM and RFGEMIE, near the carboxy terminus of the beta subunit of E . coli RNA polymerase results in a dramatic loss of the enzyme's affinity to substrates with no apparent effect on the maximal rate of the enzymatic reaction or on binding to promoters . The magnitude and selectivity of the effect suggest that the mutations disrupt the substrate binding site of the active center.

J Mol Biol, 1999 Mar 5, 286(4), 1097 - 106
Saturation mutagenesis of the E . coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair; Hortnagel K et al.; The disordered mobile loop L2 of the Escherichia coli RecA protein is known to play a central role in DNA binding and pairing . To investigate the local chemical environment in relation to function we performed saturation mutagenesis of the loop L2 region (amino acid positions 193-212) using a site-directed mutagenesis procedure, and determined the recombinational proficiency of the 380 mutants using genetic assays for homologous recombination and recombinational repair . Residues Asn193, Gln194, Arg196, Glu207, Thr209, Gly211, and Gly212 were identified as stringently required for recombinational events in bacterial cells . In addition, our findings suggest the involvement of loop L2 in the ATPase activity of RecA, and a role for residues Gln194, Arg196, Lys198 and Thr209 in the DNA-dependent hydrolysis of ATP . Finally, since 20 residue peptides that comprise this region can pair homologous DNAs by forming filamentous beta-structures, we propose how the information from the mutant analysis might facilitate the use of a simplified amino acid alphabet to design beta-structure forming L2 peptides with improved RecA-like activities .

J Mol Biol, 1999 Mar 5, 286(4), 1033 - 43
E . coli RpsO mRNA decay: RNase E processing at the beginning of the coding sequence stimulates poly(A)-dependent degradation of the mRNA; Hajnsdorf E et al.; The rpsO mRNA of E . coli encoding ribosomal protein S15 is destabilized by poly(A) tails posttranscriptionally added by poly(A)polymerase I . We demonstrate here that polyadenylation also contributes to the rapid degradation of mRNA fragments generated by RNase E . It was already known that an RNase E cleavage occurring at the M2 site, ten nucleotides downstream of the coding sequence of rpsO, removes the 3' hairpin which protects the primary transcript from the attack of polynucleotide phosphorylase and RNase II . A second RNase E processing site, referred to as M3, is now identified at the beginning of the coding sequence of rpsO which contributes together with exonucleases to the degradation of messengers processed at M2 . Cleavages at M2 and M3 give rise to mRNA fragments which are very rapidly degraded in wild-type cells . Poly(A)polymerase I contributes differently to the instability of these fragments . The M3-M2 internal fragment, generated by cleavages at M3 and M2, is much more sensitive to poly(A)-dependent degradation than the P1-M2 mRNA, which exhibits the same 3' end as M3-M2 but harbours the 5' end of the primary transcript . We conclude that 5' extremities modulate the poly(A)-dependent degradation of mRNA fragments and that the 5' cleavage by RNase E at M3 activates the chemical degradation of the rpsO mRNA .

Leukemia, 1999 Feb, 13(2), 155 - 60
Unexpected mortality from the use of E . coli L-asparaginase during remission induction therapy for childhood acute lymphoblastic leukemia: a report from the Taiwan Pediatric Oncology Group; Liang DC et al.; The relative efficacy and toxicity of E . coli L-asparaginase and epidoxorubicin used in remission induction therapy for childhood acute lymphoblastic leukemia (ALL) were assessed in a randomized trial conducted in Taiwan . All patients had standard-risk ALL, defined as a leukocyte count <10 x 10(9)/l and were aged between 1 and 2 or 7 and 10 years, or a leukocyte count <50 x 10(9)/l and were aged between 2 and 7 years, without evidence of a T cell or mature B cell immunophenotype, central nervous system leukemia or expression of two or more myeloid-associated antigens . Ninety-three patients were randomized to receive E . coli L-asparaginase at 10,000 IU/m2 thrice weekly for nine doses and 108 to receive epidoxorubicin at 20 mg/m2 weekly for two doses during remission induction with daily prednisolone, weekly vincristine and, on day 22, a dose of etoposide plus cytarabine . Patients treated with L-asparaginase had a significantly higher rate of fatal infection with or without hemorrhage than did those who received epidoxorubicin during remission induction (six of 93 vs none of 108, P = 0.009), resulting in a lower rate of complete remission in the former group (93.6 vs 99.1%, P = 0.05) . In addition, patients treated with L-asparaginase had a higher frequency of hyperglycemia and hypoalbuminemia . The overall rate of event-free survival was lower in patients treated with L-asparaginase than in other patients (P = 0.06); estimated 3-year rates were 72% (95% confidence interval, 55-89%) and 87.2% (78-96%), respectively . We conclude that L-asparaginase (Leunase) given at 10,000 IU/m2 for nine doses was poorly tolerated and resulted in excessive toxicity, both through its effects as a single agent and possibly through potentiation of etoposide.

J Mol Biol, 1999 Feb 26, 286(3), 733 - 44
Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E . coli tmRNA; Nameki N et al.; Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome . A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed . This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E . coli tmRNA tag-encoded sequence . Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants . To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance . This analysis strongly supports the presence of a pseudoknot in E . coli tmRNA, and its involvement in trans-translation . Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations . Mutations of the second stem of the pseudoknot also effect both functions . The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues . The probing data suggest the presence of magnesium ion(s) interacting with loop 2 . The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function . Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation .

J Mol Biol, 1999 Feb 19, 286(2), 417 - 35
Toxic mutations in the recA gene of E . coli prevent proper chromosome segregation; Campbell MJ et al.; The recA gene of Escherichia coli is the prototype of the recA/RAD51/DMC1/uvsX gene family of strand transferases involved in genetic recombination . In order to find mutations in the recA gene important in catalytic turnover, a genetic screen was conducted for dominant lethal mutants . Eight single amino acid substitution mutants were found to prevent proper chromosome segregation and to kill cells in the presence or absence of an inducible SOS system . All mutants catalyzed some level of recombination and constitutively stimulated LexA cleavage . The mutations occur at the monomer-monomer interface of the RecA polymer or at residues important in ATP hydrolysis, implicating these residues in catalytic turnover . Based on an analysis of the E96D mutant, a model is presented in which slow RecA-DNA dissociation prevents chromosome segregation, engendering lexA-independent, lethal filamentation of cells .

J Mol Biol, 1999 Feb 19, 286(2), 339 - 53
Conformational changes of the upstream DNA mediated by H-NS and FIS regulate E . coli RrnB P1 promoter activity; Afflerbach H et al.; The two proteins FIS and H-NS had previously been shown to regulate ribosomal RNA (rRNA) transcription by interacting with the promoter upstream DNA . FIS is known as an activator whereas H-NS had been demonstrated to function as a repressor . Details of the antagonistic control mechanisms are not yet solved . Here, we have addressed the question how the two proteins cooperate to exert both, positive and negative control of rRNA transcription . By mobility shift experiments and footprinting studies we show that FIS and H-NS binding sites partially overlap but appear to interact with different sites of a curved DNA helix . Although not mutually exclusive, the two proteins compete each other for binding . Both proteins, by changing the DNA curvature, effect circularization reactions of DNA fragments in different ways . Our results imply that binding of the proteins induces alternate DNA conformations with favourable or unfavourable topology for the formation of active transcription complexes . Together the findings presented here help to answer some of the open questions about the concerted molecular mechanism of transcription factors for the regulation of stable RNA synthesis .

Carcinogenesis, 1999 Jan, 20(1), 103 - 8
Expression of the inactive C145A mutant human O6-alkylguanine-DNA alkyltransferase in E.coli increases cell killing and mutations by N-methyl-N'-nitro-N-nitrosoguanidine; Edara S et al.; Human O6-alkylguanine-DNA alkyltransferase (AGT) counteracts the mutagenic and toxic effects of methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by removing the methyl group from O6-methylguanine lesions in DNA . The methyl group is transferred to a cysteine acceptor residue in the AGT protein, which is located at residue 145 . The C145A mutant of AGT in which this cysteine is converted to an alanine residue is therefore inactive . When this C145A mutant was expressed in an Escherichia coli strain lacking endogenous alkyltransferase activity, the number of G:C-->A:T mutations actually increased and the toxicity of the MNNG treatment was enhanced . These effects were not seen when an E.coli strain also lacking nucleotide excision repair (NER) was used . The enhancement of mutagenesis and toxicity of MNNG produced by the C145A mutant AGT was not seen with another inactive mutant Y114E that contains a mutation preventing DNA binding, and the double mutant C145A/Y114E was also ineffective . These results suggest that the C145A mutant AGT binds to O6-methylguanine lesions in DNA and prevents their repair by NER . The inactive C145A mutant AGT also increased the number of A:T-->G:C transition mutations in MNNG-treated cells . These mutations are likely to arise from the minor methylation product, O4-methylthymine . However, expression of wild-type AGT also increased the incidence of these mutations . These results support the hypothesis that mammalian AGTs bind to O4-methylthymine but repair the lesion so slowly that they effectively shield it from more efficient repair by NER.

Mol Gen Genet, 1999 Jan, 260(6), 603 - 7
Either of the chromosomal tuf genes of E . coli K-12 can be deleted without loss of cell viability; Zuurmond AM et al.; A method of lambda-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E . coli K-12 strain MG1655 . Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins . Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth . Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time . In minimal medium we observed no negative effects of tufA deletion on growth rate . Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)6 tag, into the chromosome of a strain lacking tufB.

Neoplasma, 1998, 45(5), 305 - 11
Combined therapy of B16(F10) murine melanoma using E . coli cytosine deaminase gene and murine interleukin-4 gene; Missol-Kolka E et al.; This paper summarizes preliminary results of combining suicide gene strategy (E . coli cytosine deaminase gene--CD) with immunotherapy (murine interleukin-4 gene) for treatment of experimental B16(F10) melanomas implanted into C57Bl/6 mice . The best therapeutic results, inhibition of tumor growth and prolonged survival time of treated vs . control mice, were obtained when plasmid expression vectors containing therapeutic genes were transferred into mice via DDAB/DOPE cationic liposome carrier on the third or fourth day following inoculation of mice with cancer cells . Extension of survival time has been noted in the case of two-gene therapy (as compared with one-gene therapy) of tumors which originated from cells transfected in vitro with CD gene and which were subsequently injected in vivo with IL-4-secreting cells . However, no improvement of therapeutic effect was obtained in case of mice treated with a combination of two genes transferred intratumorally with DDAB/DOPE cationic liposomes as compared to mice treated with a single gene only.

J Mol Biol, 1999 Jan 22, 285(3), 955 - 64
The kinetics of sigma subunit directed promoter recognition by E . coli RNA polymerase; Buckle M et al.; Time-resolved laser UV irradiation and controlled proteolysis have been used to study the sequential recognition of the lac UV5 promoter by Escherichia coli RNA polymerase . Local rearrangements in the DNA, the appearance of intimate protein-DNA contacts, and structural changes within the sigma subunit together provide specific signatures that define major species populated during this process . At 22 degreesC, a first closed complex is characterised by a transient conformational change in the sigma subunit and by a distortion in the -35 region . Subsequently, direct contacts at -34 and at positions -8, -5 and -3 on the non-template strand appear prior to DNA strand separation . The contact in the -35 consensus region involves only the sigma subunit . This intermediate possesses different structural parameters from that formed by quenching open complexes from 37 degreesC to 14 degreesC . Sigma thus appears as the principal partner acting during promoter recognition, a strongly coupled process involving two major intermediates only .

J Mol Biol, 1999 Jan 29, 285(4), 1667 - 77
Crystal structure of CcdB, a topoisomerase poison from E . coli; Loris R et al.; The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms . The protein consists of a five-stranded antiparallel beta-pleated sheet followed by a C-terminal alpha-helix . In one of the loops of the sheet, a second small three-stranded antiparallel beta-sheet is inserted that sticks out of the molecule as a wing . This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site . A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the alpha-helix . The surface of the dimer on the side of the alpha-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges . We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA .

Microbiology, 1998 Dec, 144 ( Pt 12), 3297 - 308
A factor produced by Escherichia coli K-12 inhibits the growth of E . coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered; Cook GM et al.; Escherichia coli produces an extracellular factor that inhibits the aerobic growth of Cyd- mutants, defective in the synthesis or assembly of the cytochrome bd-type quinol oxidase . This paper shows that such a factor is the iron-chelating siderophore enterochelin . Mutants in entA or aroB, defective in the production of enterochelin, did not produce the factor that inhibits the growth of cydAB and cydDC mutants; purified enterochelin inhibited the growth of Cyd- mutants, but not that of wild-type cells . Other iron-chelating agents, particularly ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA), whose complex with Fe(III) has a large stability constant (log K = 33.9), also inhibited the growth of Cyd- mutants at micromolar concentrations, but not that of wild-type cells . Supplementation of agar plates with Fe(III) or boiled catalase prevented the inhibition of Cyd- mutants by the extracellular factor . Spontaneous mutants isolated by being able to grow in the presence of the extracellular factor on plates also showed increased resistance to iron chelators . The reducing agent ascorbate, ascorbate plus In(III), ascorbate plus Ga(III), or Ga(III) alone, also alleviated inhibition by the extracellular factor, presumably by reducing iron to Fe(II) and complexing of the siderophore with alternative trivalent metal cations . The preferential inhibition of Cyd- mutants by the extracellular factor and other iron chelators is not due to decrease in expression, activity or assembly of cytochrome bo', the major alternative oxidase mediating quinol oxidation . Cyd- mutants overproduce siderophores, presumably reflecting intracellular iron deprivation.

J Mol Biol, 1999 Jan 15, 285(2), 843 - 55
Conserved residues in the mechanism of the E . coli Class II FBP-aldolase; Plater AR et al.; The two classes of fructose-1,6-bisphosphate aldolase both catalyse the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate . The Class I aldolases use Schiff base formation as part of their catalytic mechanism, whereas the Class II enzymes are zinc-containing metalloproteins . The mechanism of the Class II enzymes is less well understood than their Class I counterparts . We have combined sequence alignments of the Class II family of enzymes with examination of the crystal structure of the enzyme to highlight potentially important aspartate and asparagine residues in the enzyme mechanism . Asp109, Asp144, Asp288, Asp290, Asp329 and Asn286 were targeted for site-directed mutagenesis and the resulting proteins purified and characterised by steady-state kinetics using either a coupled assay system to study the overall cleavage reaction or using the hexacyanoferrate (III) oxidation of the enzyme bound intermediate carbanion to investigate partial reactions . The results showed only minor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and Asp329 mutants, suggesting that these residues play only minor or indirect roles in catalysis . By contrast, mutation of Asp109 or Asn286 caused 3000-fold and 8000-fold decreases in the kcat of the reaction, respectively . Coupled with the kinetics measured for the partial reactions the results clearly demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate . Fourier transform infra-red spectroscopy of the wild-type and mutant enzymes has further delineated the role of Asp109 as being critically involved in the polarisation of the carbonyl group of glyceraldehyde 3-phosphate .

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 16 - 24
Structure of a monoclinic crystal from of cyctochrome b1 (Bacterioferritin) from E . coli; Dautant A et al.; Crystals of E . coli cytochrome b1, alias bacterioferritin, were grown fr om a low ionic strength solution . The resulting monoclniic P21 structure was solved by molecular replacement and refined using noncrystallographi c symmetries applied to the fundamental unit, consisting of two protein subunits and a single haem . From the Patterson self-rotation results it was shown that the asymmetric unit of the monoclinic crystal consists of 12 such dimers and corresponds to a complete, nearly spherical, molecule of bacterioferritin (M4 = 450 kDa) of 432 point-group symmetry . It is thus the most symmetrical cytochrome . As previously determined for the tetragonal form, the haem is located in a special position on a local twofold axis of the dimer . A bimetal centre is also observed within the four-helix bundle of each monomer; a metal-binding site is located on the fourfold axis.

Neurol Res, 1998 Dec, 20(8), 689 - 96
Temporal profile of adenovirus-mediated E . coli lacZ gene expression in normal and post-ischemic gerbil hippocampus and ventricle; Abe K et al.; A replication-defective adenoviral vector containing the E . coli lacZ gene was directly injected into normal and post-ischemic gerbil right hippocampus and lateral ventricle, and temporal profiles of the exogenous gene expression were compared . In case of ischemia, common carotid arteries (CCA) were transiently occluded for 5 min, and the adenoviral vector was administered just after the reperfusion . The animals were recovered for 8 h, 1, 3, 7 or 21 days . A small to moderate number of neural cells in the normal hippocampus expressed the gene from 1-3 days except for the cells around dentate gyrus (DG) and the needle route that began to express from 8 h of injection . Some normal hippocampal cells persisted the expression until 7 days . A moderate to large number of ventricular cells expressed the lacZ gene from 8 h to 7 days in the normal brain . On the other hand, no expression of the lacZ gene was observed in the post-ischemic hippocampus at 8 h including cells at DG and the needle route . Hippocampal CA1 neurons, that were selectively lost at 7 days of reperfusion, never expressed the gene throughout the post-ischemic course . The other hippocampal cells such as CA3 and dentate granule cells that survived ischemia expressed the gene only transiently at 1 day . A robust expression of the gene persisted in the ventricular cells from 8 h to 7 days . The majority of brain cells in the hippocampus that expressed the lacZ gene was not the pyramidal neurons, but small neurons at around the pyramidal layers of DG . Some astroglial, but no microglial, cells expressed the lacZ gene in the hippocampus . The present study shows that an expression of the lacZ gene was limited in the post-ischemic gerbil hippocampus especially at the vulnerable CA1 layer in contrast to the strong and persistent expression in the ventricular cells, and that the majority of beta-gal positive cells were not the pyramidal neurons but small neurons at around the cell layer both in the control and post-ischemic gerbil hippocampus.

Gene, 1998 Nov 26, 223(1-2), 115 - 28
Acquired mutations in phage lambda genes O or P that enable constitutive expression of a cryptic lambdaN+cI{Ts}cro- prophage in E . coli cells shifted from 30 degreesC to 42 degreesC, accompanied by loss of immlambda and Rex+ phenotypes and emergence of a non-immune exclusion-state; Hayes S et al.; The majority of bacteria, which carry the N+-cI857{Ts}-cro--O+-P+ fragment of lambda genome, are killed when derepressed by shifting from 30 degreesC to 42 degreesC . Among rare survivors, we observed a proportion of colony-forming units (cfu) that retained the typical immlambda-immunity phenotype when grown at 30 degreesC; however, when shifted from 30 degreesC to 42 degreesC, they lost lambda immunity and acquired a non-immune exclusion-state (Nie phenotype) . We also found that the immlambda survivor cfu quickly lost their Rex+ exclusion phenotype (as measured by T4rII plating inhibition) when shifted from 30 degreesC to 42 degreesC, even though they produced CII, which stimulates pE-cI-rexA-rexB transcription . The Nie phenotype was characterized by an inhibition of plating of the homoimmune phage, lambdawt, and the heteroimmune phage, lambdaimm434 . However, lambdavir and spontaneous mutants of lambdawt (lambdase mutations localized within oR) escaped the Nie exclusion-state and plated efficiently on lawns of Nie cfu at 42 degreesC . Thus, we examined the scope of the Nie exclusion-state toward lambda mutants blocked for lysogeny, and lambda hybrids substituted for immunity or replication genes . Phage like lambdawt, competent for lysogeny, were severely excluded compared to some mutants of lambda defective for lysogeny . Among this latter type, there was high variance in the Nie exclusion of various cI mutants; some of which were not excluded . The Nie exclusion-state was attributed to the constitutive expression of the defective lambda fragment in the survivor cfu, made possible by the acquired replication defect(s) . We characterized, both genetically and physically, the mutations in the defective integrated lambda prophage that permitted growth of the survivor cfu at 42 degreesC . In five of seven survivor cfu, we identified IS2 insertions within lambda genes O and P that can block replication initiation from the lambda fragment . The remaining survivor cfu had multiple base substitutions within the C-terminal end of O and N-terminal half of P, the majority of which were silent . In some of these mutants, either an ochre nonsense mutation or a single-base frameshift deletion inactivated P.

Biophys Chem, 1998 Nov 16, 75(2), 151 - 60
Mechanism of the alpha-complementation reaction of E . coli beta-galactosidase deduced from fluorescence correlation spectroscopy measurements; Meyer-Almes FJ et al.; The kinetics of the alpha-complementation reaction of two protein fragments yielding active E . coli beta-galactosidase was measured using fluorescence correlation spectroscopy (FCS) . The association reaction was extremely slow with an apparent association rate kapp of 207 M-1 s-1 . This low association rate can be explained by a fast pre-equilibrium and slow subsequent steps involving at least two dimeric complexes . The subsequent formation of a tetrameric complex is probable and consistent with the experimental data . The complexes comprise two or four subunits, respectively, of the large fragment (EA)2 and in all cases only one small fragment, ED which has been labeled with Cy5 . These kinetics have been compared to the association kinetics of ED to inactivated (EA)2 . The kinetics were similar to the association with native (EA)2 . The data support the observation that lyophilization of (EA)2 in a reducing environment which causes complete loss of enzymatic activity does not interfere with binding.

Biophys Chem, 1998 Nov 16, 75(2), 97 - 103
Differential stability of E . coli ribosomal particles and free RNA towards thermal degradation studied by microcalorimetry; Bonincontro A et al.; We investigated the thermal degradation of E . coli ribosomes by differential scanning microcalorimetry . The 70S particles show two distinctive and irreversible peaks upon thermal degradation . Free rRNA in solution produces, on the contrary, an unstructured denaturation profile . The thermal analysis of 50S particles shows a profile substantially identical to that observed in 70S, while 30S particles produce an unstructured denaturation pattern . Therefore the thermal behavior of the 70S particle is essentially attributable to the denaturation of the 50S subunit . Our data validate previous observations that the 50S has a more rigid structure as compared to 30S, which behaves as a 'floppy' particle . In addition our data suggest that protein/RNA interactions play a significant role to stabilize three-dimensional structures of the ribosome.

EMBO J, 1998 Dec 15, 17(24), 7490 - 7
Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E . coli ribosome; Pape T et al.; The kinetic mechanism of elongation factor Tu (EF-Tu)-dependent binding of Phe-tRNAPhe to the A site of poly(U)-programmed Escherichia coli ribosomes has been established by pre-steady-state kinetic experiments . Six steps were distinguished kinetically, and their elemental rate constants were determined either by global fitting, or directly by dissociation experiments . Initial binding to the ribosome of the ternary complex EF-Tu.GTP.Phe-tRNAPhe is rapid (k1 = 110 and 60/micromM/s at 10 and 5 mM Mg2+, 20 degreesC) and readily reversible (k-1 = 25 and 30/s) . Subsequent codon recognition (k2 = 100 and 80/s) stabilizes the complex in an Mg2+-dependent manner (k-2 = 0.2 and 2/s) . It induces the GTPase conformation of EF-Tu (k3 = 500 and 55/s), instantaneously followed by GTP hydrolysis . Subsequent steps are independent of Mg2+ . The EF-Tu conformation switches from the GTP- to the GDP-bound form (k4 = 60/s), and Phe-tRNAPhe is released from EF-Tu.GDP . The accommodation of Phe-tRNAPhe in the A site (k5 = 8/s) takes place independently of EF-Tu and is followed instantaneously by peptide bond formation . The slowest step is dissociation of EF-Tu.GDP from the ribosome (k6 = 4/s) . A characteristic feature of the mechanism is the existence of two conformational rearrangements which limit the rates of the subsequent chemical steps of A-site binding.

J Med Microbiol, 1998 Dec, 47(12), 1039 - 45
New perspectives on the role of Escherichia coli O157:H7 and other enterohaemorrhagic E . coli serotypes in human disease; Goldwater PN et al.; This review compares the rates of detection of non-O157:H7 enterohaemorrhagic Escherichia coli (EHEC) with EHEC O157:H7 in outbreaks and sporadic cases of human disease by analysing Australian data and the world literature . Numerous outbreaks of disease have been attributed to EHEC O157:H7 . In many studies, isolation rates of this organism have been low and attempts to seek other EHEC have not been made . Ease of isolation and identification of the O157:H7 serotype may have given the impression that this serotype was the sole organism responsible for the outbreaks . Careful review and analysis shows that serotypes other than O157:H7 also play an important role in human disease . Evidence is presented from several overseas outbreaks described in the literature, as well as from investigations of the Adelaide O111:H- outbreak, that suggests an association between severity of disease and multiple infecting serotypes . While not diminishing the role of the O157:H7/H- clone, this review indicates that other serotypes can be responsible for outbreaks as well as cases of sporadic human disease . The current focus on O157:H7 has major implications in terms of diagnosis, the food industry and human health.

J, Mar . Biotechnol. . 1998 Aug, 6(3), 142 - 51
Physiological changes in the juvenile euryhaline teleost, the tilapia Oreochromis hornorum, injected with E . coli-derived homologous growth hormone; Guillen I I et al.; Growth is a complex process in fish . This study was designed to test the effect of different levels of recombinant tilapia growth hormone (tiGH) injected intraperitoneally in juvenile hybrid tilapia Oreochromis hornorum . Tilapia GH cDNA was cloned from hybrid O . hornorum tilapia . The mature protein was expressed in E . coli under regulation of the phage T7 promoter . The E . coli-derived tiGH was partially purified to 67% purity and, following renaturation, was shown to be biologically active in in vivo and in vitro assays . Recombinant tiGH stimulated extracellular matrix synthesis as shown by 35S-sulfate uptake in ceratobranchial cartilage explants . Zero, 0.1, 0.5 and 2.5 microg tiGH/g body weight (gbw) were injected in tilapia, and the effects on the growth-promoting action, hepatosomatic index (HSI), and mRNA insulin-like growth factor (IGF) induction were measured . A significant increase in the body weight (P < 0.05) and length (P < 0.01) was observed in tilapia receiving 0.5 microg tiGH/gbw . However, tilapia receiving 0.1 and 2.5 microg tiGH/gbw did not show an increase in body weight and length with respect to the control group receiving BSA injections . Binding sites for the recombinant tiGH were identified in the liver . Consistent with its somatotropic actions, the IGF mRNA induction was observed in the groups injected with 0.1 and 0.5 microg tiGH/gbw (P < 0.05) . No significant increase in the HSI was detected in the injected groups when compared to the control group . These results demonstrated that the injection of biologically active E . coli-derived tiGH produces physiological changes in juvenile tilapia that ultimately resulted in a growth-promoting action only at a dose of 0.5 microg tiGH/gbw.

Cell Death Differ, 1998 Nov, 5(11), 921 - 9
Rho-dependent cell spreading activated by E.coli cytotoxic necrotizing factor 1 hinders apoptosis in epithelial cells; Fiorentini C et al.; Cell-cell and cell-matrix interactions play a pivotal role in numerous cell functions including cell survival and death . In this work, we report evidence that the Rho-dependent cell spreading activated by a protein toxin from E . coli, the cytotoxic necrotizing factor 1 (CNF1), is capable of hindering apoptosis in HEp-2 cells . In addition to the promotion of cell spreading, CNF1 protects cells from the experimentally-induced rounding up and detachment and improves the ability of cells to adhere to each other and to the extracellular matrix by modulating the expression of proteins related to cell adhesion . In particular, the expression of integrins such as alpha 5, alpha 6 and alpha v, as well as of some heterotypic and homotypic adhesion-related proteins such as the Focal Adhesion Kinase, E-cadherin, alpha and beta catenins were significantly increased in cells exposed to CNF1 . Our results suggest, however, that the promotion of Rho-dependent cell spreading is the key mechanism in protecting cells against apoptosis rather than cell adhesion per se . A toxin inducing cell spreading without activating Rho, such as Cytochalasin B, was in fact ineffective in favouring cell survival . These data are of relevance (i) for the understanding of the role of the actin-dependent and especially Rho-dependent cellular activities involved in apoptosis regulation and (ii) in providing some clues to understanding the mechanisms by which bacteria, by controlling cell fate, might exert their pathogenic activity.

FEBS Lett, 1998 Nov 20, 439(3), 271 - 4
The Na+ and K+ transport deficiency of an E . coli mutant lacking the NhaA and NhaB proteins is apparent and caused by impaired osmoregulation; Verkhovskaya ML et al.; Cells of the E . coli mutant EP432, which lacks the two Na+/H+ antiporters, NhaA and NhaB, have been reported to have an impaired sodium transport activity (Harel-Bronstein et al . (1995) J . Biol . Chem . 270, 3816-3822) . Here we report that active transport of Na+ in EP432 cells can be restored to wild-type levels, either by a high K+ concentration or by an increase in the medium osmolarity . We suggest that this mutant is primarily deficient in osmoregulation rather than in cation transport per se.

Biochem Mol Biol Int, 1998 Nov, 46(4), 839 - 46
Thioredoxin fusion/HIV-1 protease coexpression system for production of soluble human IL6 in E . coli cytoplasm; Han BG et al.; In this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E . coli cytoplasm . A novel TRX gene fusion vector was developed that contained at the 3'-end of TRX gene a short DNA sequences encoding a linker peptide '-GSGSGVSQNYPIVQHHHHHH-', serving not only as a specific HIV-1 protease site but also providing six contiguous histidine (His) residues to foreign proteins . The cDNA for hIL6 was cloned into this vector resulting in plasmid pTRX@HISIL6 . The cDNA for the HIV-1 protease has been cloned into another compatible plasmid pHMM2, resulting in plasmid pHMM2-PR . Both plasmids were transformed into E . coli strain GI724, and when induced for expression of both proteins, the correct processing of TRX@HISIL6 was obtained, producing hIL6 with His6-tag at the N terminus named HISIL6 . A fraction of HISIL6 was found in soluble form and could be purified to homogeneity by Ni-NTA Superflow and ion-exchange chromatography . The biological activity of purified HISIL6 was measured by MTT method in an IL-6-dependent cell line 7TD1 to be 2.1 x 10(8) unit/mg.

Gene Expr, 1998, 7(3), 149 - 61
Overexpression of outer membrane porins in E . coli using pBluescript-derived vectors; Ghosh R et al.; The genes coding for four major outer membrane porins of Escherichia coli, ompF, ompC, phoE, and lamB, have been cloned into pBluescript-derived vectors and overexpressed to very high level (approximately 80% of the total membrane protein) in widely used host strains lacking one or more porins . For OmpF, OmpC, and PhoE porins it is shown that, contrary to current dogma, the genes can be overexpressed without undue deleterious effects upon cell growth and are stable, even under conditions of continuous expression . In contrast, overexpression of LamB is toxic to cell growth, but can be performed using tightly regulated lac promotor-driven expression . The vectors described allow overexpression, sequencing, and mutagenesis to be performed using a single system, without the necessity of subcloning, thus simplifying genetic manipulation . A particular advantage of these new vectors (with the exception of the vector for LamB) is that they do not require a particular regime for inducing the recombinant protein . To our knowledge, this study is the only comparative study of widely used membrane porin expression systems and the first to show that several porins can be stably expressed individually and maintained on high copy number vectors.

Biochemistry, 1998 Dec 1, 37(48), 17040 - 7
The different functions of BglF, the E . coli beta-glucoside permease and sensor of the bgl system, have different structural requirements; Chen Q et al.; The Escherichia coli BglF protein (EIIbgl) is an Enzyme II (EII) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) which catalyses transport and phosphorylation of beta-glucosides . In addition to its transport function, BglF serves as a beta-glucoside sensor which reversibly phosphorylates BglG, the transcription regulator of the bgl operon . Like many other PTS sugar permeases, the BglF protein is composed of three discrete functional and structural domains: IIAbgl and IIBbgl, which are hydrophilic, and IICbgl, which is hydrophobic . The domains of BglF are covalently linked to one another in the order BCA . The IIAbgl domain contains the first phosphorylation site, which accepts a phosphoryl group from the general PTS protein HPr and delivers it to the second phosphorylation site, located in the IIBbgl domain . This second site can deliver the phosphoryl group either to a beta-glucoside or to BglG . To elucidate the mechanism by which such different substrates can be phosphorylated by the same active site, we decided to try to separate the different phosphorylation activities catalyzed by BglF . To this end we rearranged the BglF domains and constructed IICBAbgl (scrambled-BglF) . Scrambled-BglF behaved like wild-type BglF in its ability to be phosphorylated and to phosphorylate BglG in vitro and in vivo . However, it could not catalyze phosphorylation of beta-glucosides in vitro nor their phosphotransfer in vivo, and it could not catalyze BglG dephosphorylation in vitro or in vivo . Therefore, the two reactions induced by beta-glucosides, sugar phosphorylation and BglG dephosphorylation, seem to require a specific domain organization: IIBbgl should precede IICbgl . The order of the B and C domains is irrelevant for BglG phosphorylation, which occurs in the absence of beta-glucosides . Because the domain order affects the way that the domains are able to interact, our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl . In light of the previous assumption that domain order in EIIs is immaterial for their function, the finding that the order of the domains is important for the function of BglF as a sugar phosphotransferase raises two possibilities: (a) BglF differs from other EIIs in this regard; (b) BglF represents a subgroup of EIIs in which the requirement for a specific domain order correlates with the ability to transport a set of structurally related sugars.

Genet Anal, 1998 Oct, 14(4), 133 - 9
Viability of E . coli cells containing phage RNA polymerase and promoter: interference of plasmid replication by transcription; Kwon YS et al.; Strong transcription of phage promoters often renders the host E . coli cells containing the phage RNA polymerase inviable . When expression of the phage SP6 RNA polymerase gene in one plasmid was induced in the E . coli JM109 cells, cells that bear an active SP6 promoter were inviable . When it was not induced (the polymerase was still produced in low levels), viability of the host cells and stability of the promoter-bearing plasmids depended on the orientation of the promoter with respect to that of the replication origin and on the sequence of the origin . A group of SP6 promoter-bearing plasmids (group I plasmids) that had the promoter directed towards the ColE1 replication origin, rendered the polymerase-containing host cells inviable in selective media . When the sequence of the origin was different (group II plasmids), this adverse effect was not observed . When the promoter direction was same as the replication origin and the ampicillin-resistant gene (group III plasmids), many satellites formed around the colonies on ampicillin-containing agar plates . These effects were caused by strong transcription of the phage SP6 promoter by its RNA polymerase, since they were reduced or eliminated by inserting an active terminator just downstream of the promoter . The viability of host cells and copy number of the promoter/terminator-bearing plasmids appear to be quantitatively related with efficiency of initiation and termination of the phage transcription . These systems may be useful for in vivo screening for mutant variants of the phage promoter, polymerase and terminator that are affected in their efficiency.

Biosens Bioelectron, 1998 Oct 1, 13(7-8), 839 - 45
Development of a biosensor for E . coli based on a flexural plate wave (FPW) transducer; Pyun JC et al.; To fulfill the need for rapid, cost-effective and sensitive methods for the detection of bacteria in medical diagnostics, food technology, biotechnology and environmental monitoring, a development of a bacterial sensor was initiated . Our approach of a biosensor for E . coli is based on an acousto-gravimetric flexural plate wave (FPW) transducer (gravimetric detection limit of less than 6 ng in a 32 microns thick sensitive layer in aqueous media), and an immunoaffinity layer on the transducer membrane for the molecular recognition of the target bacteria . An intermediate layer of covalently coupled poly (acrylic acid) yielded a major reduction of the non-specific binding to the metal surface . Such a biosensor, using antibodies against E . coli K12 and E . coli 15 outer surface antigens, yielded a detection range of 3.0 x 10(5) to 6.2 x 10(7) cells/ml for samples with the corresponding bacteria . To increase the sensitivity further, an amplification method using microspheres coupled with antibodies against E . coli was tested as a sandwich assay, and up to now a five-fold amplification of the signal has been achieved.

Cell, 1998 Nov 13, 95(4), 531 - 40
CTG repeats show bimodal amplification in E . coli; Sarkar PS et al.; Trinucleotide repeats in human genetic disorders showing anticipation follow two inheritance patterns as a function of length . Inheritance of 35-50 repeats show incremental changes, while tracts greater than 80 repeats show large saltatory expansions . We describe a bacterial system that recapitulates this striking bimodal pattern of CTG amplification . Incremental expansions predominate in CTG tracts < Okazaki fragment size, while saltatory expansions increase in repeat tracts > or = Okazaki fragment size . CTG amplification requires loss of SbcC, a protein that modulates cleavage of single-stranded DNA and degradation of duplex DNA from double-strand breaks . These results suggest that noncanonical single strand-containing secondary structures in Okazaki fragments and/or double-strand breaks in repeat tracts are intermediates in CTG amplification.

EMBO J, 1998 Nov 16, 17(22), 6487 - 96
Substrate-induced assembly of a contiguous channel for protein export from E.coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore; Thanabalu T et al.; The toxin HlyA is exported from Escherichia coli, without a periplasmic intermediate, by a type I system comprising an energized inner-membrane (IM) translocase of two proteins, HlyD and the traffic ATPase HlyB, and the outer-membrane (OM) porin-like TolC . These and the toxin substrate were expressed separately to reconstitute export and, via affinity tags on the IM proteins, cross-linked in vivo complexes were isolated before and after substrate engagement . HlyD and HlyB assembled a stable IM complex in the absence of TolC and substrate . Both engaged HlyA, inducing the IM complex to contact TolC, concomitant with conformational change in all three exporter components . The IM-OM bridge was formed primarily by HlyD, which assembled to stable IM trimers, corresponding to the OM trimers of TolC . The bridge was transient, components reverting to IM and OM states after translocation . Mutant HlyB that bound, but did not hydrolyse ATP, supported IM complex assembly, substrate recruitment and bridging, but HlyA stalled in the channel . A similar picture was evident when the HlyD C-terminus was masked . Export thus occurs via a contiguous channel which is formed, without traffic ATPase ATP hydrolysis, by substrate-induced, reversible bridging of the IM translocase to the OM export pore.

Curr Opin Biotechnol, 1998 Oct, 9(5), 497 - 501
Advances in refolding of proteins produced in E . coli; Lilie H et al.; Inclusion body production is a common theme in recombinant protein technology . Hence, renaturation of these inclusion body proteins is a field of increasing interest for gaining large amounts of proteins . Recent developments of renaturation procedures include the inhibition of aggregation during refolding by the application of low molecular weight additives and matrix-bound renaturation techniques.

Mol Gen Genet, 1998 Oct, 259(6), 645 - 55
Increased sensitivity of E . coli to novobiocin, EDTA and the anticalmodulin drug W7 following overproduction of DjlA requires a functional transmembrane domain; Bernard S et al.; In earlier studies we found that E . coli is sensitive to anticalmodulin drugs such as W7 . Mutants that are resistant to this drug were isolated, including wseA1 . In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant . We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein . It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B . In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested . Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required . the full-length, wild-type protein had a more pronounced effect . In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

Mol Gen Genet, 1998 Oct, 259(6), 610 - 4
Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E . coli; Han JS et al.; The E . coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase . We found that the IciA protein binds specifically to the AT-rich upstream region of nrd promoter . In vivo overexpression of IciA increases the expression of nrd gene by four- to five-fold, suggesting that IciA functions as a transcriptional activator for the nrd gene.

Biochem Mol Biol Int, 1998 Oct, 46(3), 479 - 86
Fusion expression of human pro-urokinase with E . coli thioredoxin; Sun AL et al.; Human pro-urokinase (pro-UK) was cloned into plasmid pET32b and fused to the E . coli thioredoxin (trxA) . When expressed in E . coli AD494(DE3), the fusion protein Trx-pro-UK accumulated as insoluble inclusion bodies and amounted to 35% of total cellular proteins . When co-expressed with molecular chaperones human protein disulfide isomerase (PDI) and E . coli GroESL, all the expressed products still existed in the form of insoluble inclusion bodies.

FEMS Microbiol Lett, 1998 Nov 1, 168(1), 71 - 5
Analysis of the length distribution of murein glycan strands in ftsZ and ftsI mutants of E . coli; Ishidate K et al.; The chain length distribution of murein glycan strands was analyzed in wild-type cells and in cells in which preseptal and/or septal murein synthesis was prevented in ftsZ84 and ftsI36 mutants of E . coli . This revealed a significant change in glycan chain lengths in newly synthesized murein associated with inactivation of the ftsZ gene product but not with inactivation of the ftsI gene product . This is the first reported abnormality in murein biosynthesis associated with mutation of an essential cell division gene.

Horm Metab Res, 1998 Sep, 30(9), 559 - 64
Recombinant IA-2 expressed in E . coli can be used for the routine detection of autoantibodies in Type-I diabetes; Morgenthaler NG et al.; We have investigated the possibility of measuring autoantibodies to IA-2 (IA-2A) using recombinant protein expressed in E . coli in a new radioassay . The intracellular part of IA-2 (IA-2ic) was expressed in E . coli as a biotinylated fusion protein and affinity-purified on a streptavidin column . The average yield of IA-2ic was about 1 mg purified protein from one litre of culture medium with E . coli . We could demonstrate the immunological activity of this material by blocking the autoantibody reactivity to in vitro synthesised IA-2ic . The IA-2ic fusion protein was then radiolabelled with 125I, purified by HPLC, and used in an immunoprecipitation assay for the detection of IA-2A . Sera from 46 of 68 (67%) patients with Type-I diabetes were positive by this radioassay, in contrst to only 2 of 50 (4%) patients with autoimmune thyroid disease and 1 of 114 (1 %) controls . There was a correlation between this radioassay and the previously established radioligand assay using synthesized 35S-methionine-labelled IA-2ic in vitro (r = 0.79, p < 0.001) . We conclude that E . coli-derived IA-2 has the correct immunogenic conformation, and can be used for the detection of IA-2A with a similar sensitivity and specificity as the validated radioligand assay . This new assay can facilitate the measurement of IA-2A in routine laboratories where the radioligand assay is inconvenient or not available.

Br J Pharmacol, 1998 Oct, 125(3), 542 - 8
Essential role for endothelin ET(B) receptors in fever induced by LPS (E . coli) in rats; Fabricio AS et al.; 1 . The influence of endothelin receptor antagonists on febrile responses to E . coli lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and endothelin-1 (ET-1) was assessed in conscious rats . 2 . Intravenous (i.v.) LPS (5.0 microg kg(-1)) markedly increased rectal temperature to a peak of 1.30 degrees C over baseline at 2.5 h . Pretreatment with the mixed endothelin ET(A)/ET(B) receptor antagonist bosentan (10 mg kg(-1), i.v.) or the selective endothelin ET(B) receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1-methoxycarboyl-D-norleucine; 3 pmol, into a lateral cerebral ventricle-i.c.v.) reduced the peak response to LPS to 0.90 and 0.75 degrees C, respectively . The selective endothelin ET(A) receptor antagonist BQ-123 (cyclo{D-Trp-D-Asp-Pro-D-Val-Leu}; 3 pmol, i.c.v.) was ineffective . 3 . Increases in temperature caused by IL-1beta (180 fmol, i.c.v.), TNF-alpha (14.4 pmol, i.c.v.) or IL-1beta (150 pmol kg(-1), i.v.) were unaffected by BQ-788 (3 pmol, i.c.v.) . 4 . Central injection of endothelin-1 (0.1 to 3 fmol, i.c.v.) caused slowly-developing and long-lasting increases in rectal temperature (starting 2 h after administration and peaking at 4-6 h between 0.90 and 1.15 degrees C) which were not clearly dose-dependent . The response to endothelin-1 (1 fmol, i.c.v.) was prevented by BQ-788, but not by BQ-123 (each at 3 pmol, i.c.v.) . Intraperitoneal pretreatment with the cyclo-oxygenase inhibitor indomethacin (2 mg kg(-1)), which partially reduced LPS-induced fever, did not modify the hyperthermic response to endothelin-1 (3 fmol, i.c.v.) . 5 . Therefore, central endothelin(s) participates importantly in the development of LPS-induced fever, via activation of a prostanoid-independent endothelin ET(B) receptor-mediated mechanism possibly not situated downstream from IL-1beta or TNF-alpha in the fever cascade.

Biochem Biophys Res Commun, 1998 Oct 20, 251(2), 509 - 14
The Zn(II) binding motifs of E . coli DNA topoisomerase I is part of a high-affinity DNA binding domain; Ahumada A et al.; Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs . Three subclones containing these tetracysteine motifs were expressed and purified . Subclone ZD1 contained the minimal tetracysteine motifs sequence . A larger subclone ZD2 corresponded to a region bordered by two protease sensitive sites . Subclone ZD3 also included the 14-kDa C-terminal domain that has been shown to bind DNA . Subclones ZD1 and ZD2 were found to bind one and two Zn(II), respectively, and neither had detectable DNA binding activity . ZD3 could bind three Zn(II) and had higher DNA binding affinity than the 14-kDa C-terminal domain . The complex formed between ZD3 and a single-stranded 31mer could be detected by the gel shift assay while the complex formed by the 14-kDa C-terminal domain was not stable under gel electrophoresis conditions . The three Zn(II) binding motifs appeared to be part of a high-affinity DNA binding domain .

Neurosci Lett, 1998 May 1, 246(3), 153 - 6
Expression of adenovirus-mediated E . coli lacZ gene in skeletal muscles and spinal motor neurons of transgenic mice with a mutant superoxide dismutase gene; Warita H et al.; A replication-defective recombinant adenoviral vector containing E . coli lacZ gene was injected into the right biceps brachii muscles of transgenic mice carrying mutant human Cu/Zn superoxide dismutase (SOD1) gene and non-transgenic wild-type mice at 27 weeks of age . Although the transgenic mice showed remarkable neurogenic muscular changes and a marked motor neuron loss in the anterior horn of spinal cord, the lacZ gene was widely expressed in all the injected muscles of transgenic mice as well as of wild-type mice at 7 days after the injection . In one transgenic and two wild-type mice, the lacZ gene expression was first detected in a few motor neurons of right lower cervical cord (C5-C6) . These results demonstrate that an adenovirus-mediated foreign gene is transferred and expressed in skeletal muscles both of normal and transgenic mice model for familial amyotrophic lateral sclerosis (FALS), and also, in the spinal motor neurons, may be transferred by retrograde transport from innervated muscles.

Braz J Med Biol Res, 1998 Aug, 31(8), 1019 - 34
E . coli alpha-hemolysin: a membrane-active protein toxin; Goni FM et al.; alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation . A second activation step takes place in the extracellular medium through binding of Ca2+ ions . Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation . However, Ca(2+)-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes . The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can alpha-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues . Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension . Protein or glycoprotein receptors for alpha-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Mol Cell, 1998 Sep, 2(3), 373 - 81
Migration of a Holliday junction through a nucleosome directed by the E . coli RuvAB motor protein; Grigoriev M et al.; Chromatin plays a critical role in regulating access to DNA by proteins that direct recombination and repair . The E . coli RuvAB protein complex promotes branch migration of the Holliday junction recombination intermediate . The ability of RuvAB to negotiate passage of the junction through nucleosomal DNA is examined . The model system involves the formation of a Holliday junction positioned upstream of a nucleosome . Unassisted, the junction is blocked by a histone octamer . In the presence of RuvAB and ATP, rapid branch migration through the nucleosome is observed . It results in disruption of the histone-DNA interactions leading to the removal of the octamer from the junction intermediate . These results suggest that eukaryotic DNA motor proteins analogous to RuvAB could function during recombination to promote branch migration through chromatin.

IMA J Math Appl Med Biol, 1998 Sep, 15(3), 257 - 78
Numerical techniques and mathematical modelling for CI857-controlled gene expression and cell growth in recombinant E . coli; Cubarsi R et al.; Recombinant gene expression, monitored by beta-galactosidase activity, is studied in a pL, pR-CI857 plasmid expression system in temperature-induced E . coli batch cultures . The experimental procedure has been mathematically modelled, and the corresponding parameters are estimated from specific statistical and numerical methods, basically by using a global least-squares procedure under some constraints induced by the model . The numerical techniques proposed in this work act by accumulation of data coming from several runs of the modelled experiment, so that more accuracy is obtained in the parameter estimation . In particular, for the production process, an extra-model parameter depending on an indicator vector is introduced for each run of the experiment in order to globalize the data . The analysis of the data obtained leads to an integrated model for both cell growth and gene expression, which describes an asymmetric dynamics between culture growth and recombinant protein yield, and can serve to predict the maximal value of accumulated gene expression and the time required for it to be achieved at any age of the preinducing cell growth.

Zhonghua Yi Xue Za Zhi, 1997 May, 77(5), 340 - 3
{A recombinant autoantigen U 1 RNP 70,000: expression in E . coli . and its serodiagnostic application}; Xue F et al.; OBJECTIVE: To express the recombinant autoantigen U 1 RNP 70,000 and to evaluate its serodiagnostic value . METHOD: We used molecular cloning techniques and immunoblotting technique . RESULTS: The recombinant proteins had authentic U 1 RNP 70,000 antigenicity, with the molecular weight corresponding to their theoretical value . 112 of 115(97.4%) anti-70,000 positive sera(IBT) could be detected by r 70,000 . CONCLUSIONS: r 70,000 has good specificity and can be used in further detailed epitopes mapping . r 70,000 can be put into use for serodiagnostic detection.

Virology, 1998 Oct 10, 250(1), 1 - 8
Hepatitis C virus E1 protein induces modification of membrane permeability in E . coli cells; Ciccaglione AR et al.; The E1 gene of hepatitis C virus (HCV) has been cloned and expressed in BL21(DE3)pLys Escherichia coli strain by pET3a vector to analyze changes in membrane permeability produced by this protein . We showed that the expression of E1 (aa 192-383), as well as of two C-terminal fragments (aa 331-383 and aa 341-383) corresponding to the transmembrane (TM) region of this protein, induced a rapid lysis of cells . On the contrary, the expression of a mutant of E1 (aa 192-340), lacking the last 40 amino acids, did not cause cell lysis . The analysis of permeability changes revealed that modification of membrane permeability to several compounds were observed only in clones expressing E1 and C-terminal fragments, while the synthesis of the C-terminal-deleted mutant had little or no effect on permeability . These findings demonstrate that the TM domain of E1 protein has membrane-active properties that may be involved in some aspects of virus-cell interaction .

Leukemia, 1998 Oct, 12(10), 1527 - 33
Anti-asparaginase antibodies following E . coli asparaginase therapy in pediatric acute lymphoblastic leukemia; Woo MH et al.; Asparaginase is an effective antileukemic agent and is included in most front-line protocols for pediatric acute lymphoblastic leukemia (ALL) worldwide; however, allergic reactions to asparaginase may be dose-limiting . We evaluated plasma anti-asparaginase antibody concentrations in a cohort of children with newly diagnosed ALL, who did and who did not exhibit clinical hypersensitivity, after Escherichia coli (E . coli) asparaginase therapy . Thirty-five children who received asparaginase 10000 IU/m2 i.m . three times weekly for nine doses as part of both multiagent induction and reinduction chemotherapy, and seven monthly doses during the first 7 months of continuation treatment, were studied . Twenty-two patients experienced initial allergic reactions to asparaginase during continuation (n=20) or reinduction (n=2) phases and 13 children did not exhibit any reaction . An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-asparaginase antibodies in plasma samples, diluted 1:3200, using E . coli asparaginase as the antigen . The median anti-asparaginase antibody concentration (OD at 1:3200 dilution) increased from 0.039 at induction to 0.506 at reinduction in patients who exhibited clinical hypersensitivity (P = 0.0002) . By comparison, median antibody level increased from 0.011 to 0.032 OD at identical time points in patients who did not react to asparaginase (P = 0.02) . Both post-induction and post-reinduction anti-asparaginase antibody levels were higher in reacting than in nonreacting patients (P = 0.004 and P = 0.01, respectively) . Antibody levels were inversely related to the time elapsed between the reaction and sampling (P = 0.011) . Although anti-asparaginase antibody levels increased from the post-induction plasma sample to the post-reinduction sample in 28 of 35 patients regardless of whether they exhibited clinical hypersensitivity, patients with hypersensitivity reactions had higher antibody levels than did identically treated control patients at comparable time points in therapy . Therefore, antibody analysis may be of clinical value in predicting future hypersensitivity.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 438 - 40
Crystallization and preliminary X-ray diffraction studies of E . coli porphobilinogen synthase and its heavy-atom derivatives; Shimoni-Livny L et al.; Porphobilinogen synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form porphobilinogen . E . coli PBGS is an homooctameric enzyme . The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion . Diffraction-quality crystals of native E . coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 A) are reported . These crystals diffract to about 3.0 A resolution.

Acta Crystallogr D Biol Crystallogr, 1998 Mar 1, 54 ( Pt 2), 281 - 3
Crystallization and preliminary X-ray diffraction studies of phospho-adenylylsulfate (PAPS) reductase from E . coli; Montoya G et al.; PAPS reductase from E . coli is involved in sulfur metabolism and catalyses the reduction of phospho-adenylyl-sulfate (PAPS) to sulfite . The protein has been cloned, overexpressed and purified from E . coli . Crystallization experiments resulted in crystals suitable for X-ray diffraction . The crystals belong to the orthorhombic space group C2221 with cell dimensions a = 81.9, b = 97.4, c = 109.5 A, and contain one molecule per asymmetric unit . At cryogenic (100 K) temperatures the crystals diffract to a resolution limit of 2.7 A using a rotating anode and to 2.0 A at a synchrotron source.

Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 684 - 6
Preliminary crystallographic investigations of recombinant GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from E . coli; Tonetti M et al.; The GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GM_ER) isolated from E . coli has been overexpressed as a GST-fusion protein and purified to homogeneity . The enzyme, an NADP+(H)-binding homodimer of 70 kDa, is responsible for the production of GDP-L-fucose . GM_ER shows significant structural homology to the human erythrocyte protein FX, which is involved in blood-group glycoconjugate biosynthesis, displaying 3,5 epimerase/reductase activity on GDP-4-keto-6-deoxy-D-mannose . GM_ER has been crystallized in a trigonal crystalline form, containing one molecule per asymmetric unit, suitable for high-resolution crystallographic investigations.

FEBS Lett, 1998 Sep 18, 435(2-3), 204 - 6
cDNA cloning of Brassica napus malonyl-CoA:ACP transacylase (MCAT) (fab D) and complementation of an E . coli MCAT mutant; Simon JW et al.; The GenBank database was searched using the E . coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated . A 272-bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E . coli MCAT . A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library . This resulted in the isolation of a 1200-bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids . The protein shows 47% homology to the E . coli MCAT amino acid sequence in the coding region for the mature protein . Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E . coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA . This is the first functional evidence supporting the identification of a plant cDNA for MCAT.

J Steroid Biochem Mol Biol, 1998 Sep, 66(5-6), 355 - 63
Evolution of mammalian 11beta- and 17beta-hydroxysteroid dehydrogenases-type 2 and retinol dehydrogenases from ancestors in Caenorhabditis elegans and evidence for horizontal transfer of a eukaryote dehydrogenase to E . coli; Baker ME; Physiological responses due to steroid hormones and retinoids are regulated by their cognate receptors and dehydrogenases . The origins of either regulatory mechanism are not fully understood . Here we examine the origins of the human 11beta-hydroxysteroid dehydrogenase-type 2, which regulates access of glucocorticoids to cells, and 17beta-hydroxysteroid dehydrogenase-type 2, which regulates access of androgens and estrogens to cells . Sequence comparisons trace their ancestry to homologs in Caenorhabditis elegans . These C . elegans proteins most closely resemble mammalian all-trans and 11-cis-retinol dehydrogenases . The similarity is sufficient -37% to 43% identity to suggest that one or more of the C . elegans homologs metabolizes a retinoid . Receptors for retinoids, but not for androgens, estrogens or glucocorticoids have been identified in C . elegans, suggesting that retinoid-mediated gene transcription is more ancient than that for adrenal and sex steroids . We propose that the hydroxysteroid dehydrogenase-type 2 mechanism for regulating the androgen, estrogen and glucocorticoid concentrations in mammals descended from that for regulating retinoid concentrations . Interestingly, E . coli contains a protein with strong sequence similarity to mammalian retinol dehydrogenases . Sequence comparisons and phylogenetic analysis indicate that the E . coli protein may be an example of horizontal transfer from a eukaryote ancestor.

J Muscle Res Cell Motil, 1998 Aug, 19(6), 639 - 46
Generation of functional beta-actinin (CapZ) in an E . coli expression system; Soeno Y et al.; beta-actinin (CapZ) is a heterodimeric actin-binding protein which caps the barbed end of action filaments and nucleates actin-polymerization in a Ca2+ -independent manner . In myofibrils it is localized in the Z-lines . As judged by these properties of b-actinin, it is conceivable that beta-actinin is involved in the regulation of actin assembly, especially in the formation of I-Z-I complex during myofribrillogenesis . In this study, we devised a system to produce functional beta-actinin in E . Coli . The cDNAs of beta I' and beta II subunits of beta-actinin were obtained by RT-PCR methods using the published sequence as references, and subcloned in a pET vector . When the proteins were produced with the cDNA of either beta I' and beta II in E . coli, the proteins were insoluble and non-functional . However, when the cDNAs encoding the two subunits were cloned into a single vector and both proteins were expressed simultaneously, the proteins became soluble and purified as a functional heterodimer The activity of the purified proteins was not distinguishable from that of beta-actinin purified from skeletal muscle.

J Mol Biol, 1998 Sep 18, 282(2), 227 - 39
Nascent RNA in transcription complexes interacts with CspE, a small protein in E . coli implicated in chromatin condensation; Hanna MM et al.; Proteins in a partially fractionated Escherichia coli extract that interact with the nascent RNA in active transcription complexes from several promoters were detected using the photocrosslinking ribonucleotide analogs 5-(azidophenacyl)thio-UTP or 5-(azidophenacyl)thio-CTP as transcription substrates . Upon irradiation of ternary transcription complexes, several extract proteins were crosslinked to the RNA . Most notably, a small protein was crosslinked to the RNA in complexes on seven of nine templates tested . This protein was purified and sequenced and found to match a hypothetical protein, MsmC/CspE, recently shown to be involved in chromatin partitioning . CspE has 69% amino acid sequence identity with the major cold shock protein in E . coli, CspA, which has been shown to bind to a DNA sequence designated the Y box, with the sequence 5'-ATTGG . Of the nine templates tested, CspE was found to be most heavily crosslinked to RNA from the lambda PR' promoter, which is modified by the Q antiterminator protein . CspE was very heavily crosslinked to RNA only ten nucleotides long in initial ternary complexes on this promoter, but not to this same RNA after it had been released from the transcription complex . However, even when present from the start of transcription, CspE did not crosslink to the RNA 82 nucleotides long in elongation complexes from this same promoter . Despite the loss of interaction with the RNA after polymerase had left the promoter, CspE inhibited Q-mediated transcriptional antitermination from PR' in vitro almost 200 nucleotides downstream from the promoter, presumably by interaction with the Y box DNA upstream from PR', which overlaps with the binding site for the Q . A potential role for CspE and transcription in chromosome condensation and nucleoid structure is discussed .

Yakugaku Zasshi, 1998 Jul, 118(7), 257 - 71
{Function of DnaA protein, the initiator for chromosomal DNA replication in E . coli}; Mizushima T; DnaA protein is an initiator for chromosomal DNA replication in E . coli . We have examined the function of the protein to answer the following four questions; 1 . How DnaA protein is inactivated after DNA replication for the suppression of re-initiation? 2 . How DnaA protein is activated for the initiation of DNA replication? 3 . Does DnaA protein have functions other than that for DNA replication? 4 . Is DnaA protein is a good target for new antibiotics? In this review, I summarize our recent studies for these questions.

Proteins, 1998 Aug 1, 32(2), 200 - 10
Effects of the T-->R transition on the electrostatic properties of E . coli aspartate transcarbamylase; Hariharan M et al.; Aspartate transcarbamylase is a large (310 kD), multisubunit protein that binds substrates cooperatively and undergoes a large change in quaternary structure when substrates bind . The forces that drive this transition are poorly understood . We evaluated the electrostatic component of these forces by using finite difference and multigrid methods to solve the nonlinear Poisson-Boltzmann equation for complexes of the enzyme with several substrates and substrate analogs . The results have been compared with calculations for the unliganded protein . While pK1/2 values of most ionizable residues fall within 3 pH units of values for model compounds, 31 have pK1/2 values that fall outside the range 0-17 . Many of these residues are at the active site, where they interact with the highly charged substrate, in the 80s loop or 240s loop or interact with these loops . The pK1/2 values of eight ionizable residues related by the twofold molecular axes differ by more than 3 pH units, providing additional evidence for asymmetry within the crystal . As in the unliganded structure, a set of residues forms a network in which ionizable groups with Wij values greater than 2 kcal-m(-1) are separated by distances greater than 5 A . Some residues participate in this network in both the unliganded and N-phosphonacetyl-L-aspartate (PALA)-liganded structure, while others are found in only one structure . The network is more extensive in the PALA-liganded structure than in the unliganded structure, but consists of two separate networks in the two halves of the molecule.

Vaccine, 1998 Oct, 16(17), 1611 - 9
Display of an inhibin epitope in a surface-exposed loop of the E . coli heat-labile enterotoxin B subunit; Sewani CR et al.; In vitro gene manipulation was used to develop a novel chimeric antigen consisting of the non-toxic B subunit (EtxB) of an E . coli enterotoxin and the first 14 N-terminal amino acid residues of the carboxy-terminal portion of the alpha subunit of bovine inhibin (bINH1-14) . Rabbits immunized subcutaneously (s.c.) or intravenously (i.v.) with EtxB::bINH1-14, with or without Freund's adjuvant, developed significant titres of antibodies that recognized an inhibin peptide fragment containing bINH1-14, native inhibins, and EtxB during separate enzyme-linked immunosorbent assay (ELISA) . Passive immunization of mice with the rabbit anti-EtxB::bINH1-14 serum increased concentrations of follicle-stimulating hormone (FSH) in serum twofold compared with controls, whereas serum concentrations of luteinizing hormone (LH) were unaltered . Since FSH is the primary hormone from the pituitary gland that stimulates ovarian follicle growth and spermatogenesis, the results of this study demonstrate that EtxB::bINH1-14 has potential as antigen for development of inhibin-based fertility vaccines.

Mutat Res, 1998 Jul 8, 415(1-2), 139 - 50
Mutagenicity of p-aminophenol in E . coli WP2uvrA/pKM101 and its relevance to oxidative DNA damage; Yoshida R et al.; It was recently reported that p-aminophenol (p-AP) induces DNA cleavage in mouse lymphoma cells, CHO cells and human lymphoblastoid cells . The mutagenicity of p-AP has not, however, been detected by reverse mutation assays . The purpose of this study was to assess the mutagenicity of p-AP by reverse mutation assay using Escherichia coli WP2uvrA/pKM101, which has a spectrum for detecting mutations different from those of other strains in the family with an AT base pair at the mutation site and has higher sensitivity to certain oxidative mutagens as compared to other strains . We found that p-AP was mutagenic to E . coli WP2uvrA/pKM101 . The mutagenic activity of this compound was suppressed with the addition of dimethylsulfoxide or catalase, suggesting the involvement of active oxygen species in the mutagenic process induced by p-AP . To further elucidate the underlying mechanism, we used isolated DNA for the following experiments . It was revealed, by gel electrophoretic analysis, that p-AP induced DNA cleavage in the presence of Fe(III) . However, p-AP alone did not induce this cleavage . Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by p-AP in calf thymus DNA was also detected in the presence of Fe(III) by HPLC with an electrochemical detector . ESR-spin trapping experiments using DMPO detected the production of hydroxyl radical (.OH) in the solution of p-AP with Fe(III) . Both p-AP mediated DNA damages and .OH production by p-AP in the presence of Fe(III) were completely inhibited by .OH scavengers (ethanol, mannitol, sodium formate, dimethylsulfoxide) and catalase . These results suggest that .OH derived from the reaction between H2O2 and Fe(III) (Fenton reaction) participates in the oxidative DNA damage . Accordingly, the same mechanism might be working in E . coli WP2uvrA/pKM101 during induction of the mutation by p-AP.

J Food Prot, 1998 Apr, 61(4), 444 - 9
Comparison of the SimPlate coliform and Escherichia coli test with Petrifilm, three-tube MPN, and VRBA + MUG methods for enumerating coliforms and E . coli in food; Townsend DE et al.; SimPlate for coliforms and Escherichia coli (CEc) is a new method for the detection and quantification of coliforms and E . coli in food . Internal validation of the method was carried out at IDEXX Laboratories (Westbrook, ME) with 180 food samples representing a variety of different food matrices and compared against three-tube MPN (most probable number), VRBA (violet red bile agar) + MUG, and Petrifilm (E . coli count) methods . SimPlate CEc was highly correlated with each of these methods for the quantification of coliform bacteria (r > or = 0.90) . An insignificant number of food samples were found to contain E . coli; therefore, no meaningful correlation data could be generated . Four hundred forty-four additional food samples were tested at five collaborating laboratories for the presence of coliforms E . coli using SimPlate CEc and either VRBA + MUG or Petrifilm (E . coli count) . Regression analysis of data from SimPlate for CEc versus Petrifilm E . coli count plates generated correlation coefficients (r) of at least 0.89 for total coliforms and at least 0.90 for generic E . coli . Correlation coefficients between SimPlate for CEc and VRBA + MUG data were at least 0.90 for coliforms and at least 0.86 for E . coli . SimPlate for CEc demonstrated better recovery of E . coli than Petrifilm when high populations of bacteria were present . E . coli was not detected in 20 or 50 (40%) raw milk samples tested by the Petrifilm method due to the presence of interfering coliform and noncoliform bacteria . It is concluded that SimPlate for CEc is a suitable alternative for determining numbers of coliform bacteria and E . coli in food.

Nucleosides Nucleotides, 1998 Jan-Mar, 17(1-3), 327 - 38
The chemical synthesis of E . coli tRNA(Lys) anticodon loop fragment and its analogues; Sochacka E; E . coli tRNA(Lys) anticodon loop fragment (Umnm5s2UUUt6A) 1 and its analogues 2-6 were synthesized by the classical phosphotriester approach in solution . The preparation of suitably protected derivatives of N6-threonylcarbamoyladenosine 18 is also described.

EMBO J, 1998 Aug 17, 17(16), 4818 - 28
Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli; Hesterkamp T et al.; Folding of newly synthesized cytosolic proteins has been proposed to require assistance by Hsp70 chaperones . We investigated whether two Hsp70 homologs of Escherichia coli, DnaK and HscA, have this role in vivo . Double mutants lacking dnaK and hscA were viable and lacked defects in protein folding at intermediate temperature . After heat shock, a subpopulation of pre-existing proteins slowly aggregated in mutants lacking DnaK, but not HscA, whereas the bulk of newly synthesized proteins displayed wild-type solubility . For thermolabile firefly luciferase, DnaK was dispensable for de novo folding at 30 degrees C, but essential for aggregation prevention during heat shock and subsequent refolding . DnaK and HscA are thus not strictly essential for folding of newly synthesized proteins . DnaK instead has functions in refolding of misfolded proteins that are essential under stress.

Toxicol Lett, 1998 May, 95(3), 147 - 54
Testing of SOS induction of artificial polycyclic musk fragrances in E . coli PQ37 (SOS chromotest); Mersch-Sundermann V et al.; Synthetic fragrances are widespread in the environment . Residues were found in animals, human tissues and breast milk . Therefore, six artificial polycyclic musk fragrances--Galaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and Traseolide--were tested for SOS induction using the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) in the presence (+S9) and absence (-S9) of an exogenous metabolizing system . All compounds tested exhibited no SOS inducing potency with the SOS chromotest . These results could be rated as one indicator of the biological inactivity of this group of compounds with respect to genotoxicity.

Cytokine, 1998 Jul, 10(7), 544 - 8
Profiles of circulating inflammatory- and anti-inflammatory cytokines in patients with hemolytic uremic syndrome due to E . coli O157 infection; Murata A et al.; The systemic inflammatory response to Escherichia coli O157 infection was studied from the profiles of circulating inflammatory and anti-inflammatory cytokines . Twelve patients transferred sequentially to our hospital for the intensive care with acute illness due to Escherichia coli O157 infection and the possible form of haemolytic uraemic syndrome were included in this study . Increased circulating concentrations of tumour necrosis factor, interleukin 6, interleukin 8, granulocyte colony-stimulating factor, and interleukin 10 were found in patients with various stages of this infection and haemolytic uraemic syndrome . Especially, the degree of the increase of circulating interleukin 10 in those who had a typical signs of haemolytic uraemic syndrome was higher than those of other inflammatory cytokines . Two groups of E . coli infection could be classified into one with a typical haemolytic uraemic syndrome and the other with atypically bacteremic state over haemolytic uraemic syndrome according to these cytokine levels.

Toxicon, 1998 Aug, 36(8), 1155 - 63
Diversity of cDNAs encoding phospholipase A2 from Agkistrodon halys pallas venom, and its expression in E . coli; Pan H et al.; As a step toward understanding the structure and function of phospholipase A2(PLA2), we isolated several novel cDNAs encoding Agkistrodon halys Pallas PLA2 isoenzymes including B-PLA2, Asn49-PLA2, A-PLA2, A'-PLA2 and BA1-PLA2 by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminus of these enzymes . The amino acid sequences of A-PLA2 deduced from cDNA are consistent with that isolated from venom except for four residues . Asn49-PLA2 and B-PLA2 are highly similar (> 95%), but the critical residue Asp49 in the active centre of B-PLA2 is replaced by Asn49 in Asn49-PLA2 . The N-terminal residues (1-24) of BA1-PLA2 shows high similarity to that of B-PLA2 which has strong ability to hemolyze erythrocytes, while its C-terminal residues (72-125) are the same as that of A-PLA2 which can inhibit platelet aggregation . The successful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA2s, but also to disclose the genesis of structural diversity of PLA2s, namely DNA modification and gene rearrangement . The cloned cDNA for A-PLA2 has been expressed in E . coli . By Q-Sepharose column chromatography, denaturation-renaturation and FPLC, we obtained the active recombinant protein with the initiator Met . This is the first report of the production of an active recombinant PLA2 with the initiator Met.

Vet Q, 1998, 20 Suppl 3, S87 - 9
Prevention of diarrhoea using pathogen specific monoclonal antibodies in an experimental enterotoxigenic E . coli infection in germfree piglets; de Geus B et al.; In the present study we describe the effect of oral application of mAB specific for ETEC F4ac fimbriae in an experimental ETEC challenge model in neonatal germfree piglets . The results show that mAB, specific for different F4ac epitopes protect animals against ETEC specific pathology . Moreover, the results show that protection is independent of F4ac epitope specificity.

Mutat Res, 1998 May 25, 400(1-2), 127 - 33
A fragment of the yeast DNA repair protein Rad4 confers toxicity to E . coli and is required for its interaction with Rad7 protein; Wei S et al.; The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair . Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli . In this study, a rad4 mutant that can be grown in E . coli was isolated . This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E . coli when expressed alone . The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair . The smallest Rad4 fragment that is toxic to E . coli consists of 336 amino acids with a calculated pI=9.99 .

Vaccine, 1998 Aug, 16(13), 1283 - 9
Immunization with E . coli produced recombinant T . gondii SAG1 with alum as adjuvant protect mice against lethal infection with Toxoplasma gondii; Petersen E et al.; Polyclonal rabbit antibodies against recombinant Toxoplasma gondii SAG1 antigen expressed in E.coli recognize T . gondii and the antibodies significantly reduced T.gondii adherence and/or invasion into the host cell as did a monoclonal antibody against a conformational epitope of the SAG1 antigen . Groups of outbread NMRI mice were immunized with recombinant T . gondii SAG1 antigen in alum . The antibody response to immunizations was dominated by a Th2 response with production of T.gondii specific IgG1 antibodies . Challenge with tachyzoites from the virulent RH-strain produced a Th1 response dominated by the production of specific IgG2a antibodies and moderately boosted the IgG1 response, and challenge with bradyzoites from the avirulent SSI119-strain showed the same pattern . Immunization with rSAG1 resulted in a significant increased survival after challenge with tachyzoites of the RH-strain . Immunization with E.coli expressed recombinant SAG1 in alum induce partial protective immunity against lethal infection with T . gondii in mice.

Mutat Res, 1998 Jun 18, 402(1-2), 59 - 66
DNA mutagenesis and repair in UV-irradiated E . coli K-12 under condition of mutation frequency decline; Fabisiewicz A et al.; This paper shows that mutation frequency decline (MFD) occurs in UV-irradiated and transiently starved Escherichia coli K-12 strain AB1157 . This effect involves preferential repair of pre-mutagenic lesions situated on the transcribed strand, and is mfd-, and uvrA-dependent . Mutations were tested by measuring reversion of argE3(ochre) to Arg+ and the types of reversions and the mutational specificity were defined . UV-irradiation induces reversions to Arg+ predominantly by forming ochre suppressors, supB and, much more rarely, supE(ochre) . In irradiated and then transiently starved cells, the ratio of supB/supE(ochre) is reversed, and supE(ochre) suppressors predominate . Back reversions at the argE3 site occurs only in bacteria defective in UvrABC-excinuclease . The introduction of a Tn10 transposon makes bacteria more sensitive to UV-irradiation and diminishes the frequency of reversions . Transformation with pGW2123, a umuD'C-bearing plasmid, recovers and enhances the frequency of reversion but has no effect on post-UV-survival of the bacteria .

Cell, 1998 Jul 10, 94(1), 61 - 71
The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E . coli chromosomal replicase; Katayama T et al.; The beta subunit of DNA polymerase III is essential for negative regulation of the initiator protein, DnaA . DnaA inactivation occurs through accelerated hydrolysis of ATP bound to DnaA; the resulting ADP-DnaA fails to initiate replication . The ability of beta subunit to promote DnaA inactivation depends on its assembly as a sliding clamp on DNA and must be accompanied by a partially purified factor, IdaB protein . DnaA inactivation in the presence of IdaB and DNA polymerase III is further stimulated by DNA synthesis, indicating close linkage between initiator inactivation and replication . In vivo, DnaA predominantly takes on the ADP form in a beta subunit-dependent manner . Thus, the initiator is negatively regulated by action of the replicase, a mechanism that may be key to effective control of the replication cycle.

Am J Gastroenterol, 1998 Jul, 93(7), 1055 - 9
The immunohistological diagnosis of E . coli O157:H7 colitis: possible association with colonic ischemia; Su C et al.; OBJECTIVE: E . coli O157:H7 may cause hemorrhagic colitis resembling ischemic colitis . Diagnosis is usually made by finding sorbitol-negative colonies on MacConkey agar that react with O157 and H7 antisera . Most ischemic colitis is idiopathic, but some may be caused by E . coli O157:H7, inasmuch as this organism can produce fibrin thrombi in colon vasculature . The objectives of this study were to determine whether E . coli O157:H7 infection can be diagnosed retrospectively from paraffin blocks of colon sections and whether an association exists between E . coli O157:H7 infection and colonic ischemia . METHODS: Paraffin-embedded sections of normal colon (n = 2) and various colitides {ischemic (n = 11), E . coli O157:H7 (n = 2), IBD (n = 8) and pseudomembranous (n = 3)} were used . Sections were deparaffinized, rehydrated, incubated with 3% peroxide in methanol, rinsed, and incubated with peroxidase-labeled antibody isolated from goats immunized with whole E . coli O157:H7 . Sections were stained with peroxidase chromagen reagent and counterstained with hematoxylin . Coarse, granular, orange-brown staining was considered positive . To determine the localization of the chromagen deposits, three cases that stained positive, including one of the culture-proved E . coli O157:H7 colitis and two of colonic ischemia, were processed for electron microscopy . RESULTS: Both cases (100%) of E . coli O157:H7 colitis and three of 11 (27.3%) cases of ischemic colitis stained positive by light microscopy . In one culture-proved case, electron microscopy demonstrated staining of bacillary structures; in two cases of colonic ischemia, extensive deposits of chromagen material were present that were associated neither with inflammatory cells nor with bacterial forms . CONCLUSIONS: Immunoperoxidase staining of archival sections may be used to diagnose E . coli O157:H7 infection . An etiological role for this organism is possible in some cases of colonic ischemia.

Bioelectromagnetics, 1998, 19(5), 300 - 9
Cell density dependent response of E . coli cells to weak ELF magnetic fields; Belyaev IYa et al.; The effects of weak magnetic fields of extremely low frequency (ELF) on E . coli K12 AB1157 cells were studied by the method of anomalous viscosity time dependencies (AVTD) . E . coli cells at different densities within a range of 5 x 10(5)-10(9) cell/ml were exposed to ELF (sinusoidal, 30 microT peak, 15 min) at a frequency of 9 Hz . A transient effect with maximum 40-120 min after exposure was observed . Kinetics of the per-cell-normalised ELF effects fitted well to a Gaussian distribution for all densities during exposure . A maximum value of these kinetics and a time for this maximum were strongly dependent on the cell density during exposure . These data suggest a cell-to-cell interaction during response to ELF . Both dependencies had three regions close to a plateau within the ranges of 3 x 10(5) - 2 x 10(7) cell/ml, 4 x 10(7) - 2 x 10(8) cell/ml and 4 x 10(8)-10(9) cell/ml and two rather sharp transitions between these plateaus . The effect reached a maximum value at a density of 4 x 10(8) cell/ml . Practically no effect was observed at the lowest density of 3 x 10(5) cell/ml . The data suggested that the ELF effect was mainly caused by a secondary rather than a primary reaction . The filtrates from exposed cells neither induced significant AVTD changes in unexposed cells nor increased the ELF effect when were added to cells before exposure . The data did not provide evidence for significant contribution of stable chemical messengers, but some unstable compounds such as radicals could be involved in the mechanism of cell-to-cell interaction during response to ELF . The results obtained were also in accordance with a model based on an re-emission of secondary photons during resonance fluorescence.

Biochemistry (Mosc), 1998 Jun, 63(6), 671 - 84
Three-dimensional structures of mutant forms of E . coli inorganic pyrophosphatase with Asp-->Asn single substitution in positions 42, 65, 70, and 97; Avaeva SM et al.; The three-dimensional structures of four mutant E . coli inorganic pyrophosphatases (PPases) with single Asp-->Asn substitutions at positions 42, 65, 70, and 97 were solved at 1.95, 2.15, 2.10, and 2.20 A resolution, respectively . Asp-42-->Asn and Asp-65-->Asn mutant PPases were prepared as complexes with sulfate--a structural analog of phosphate, the product of enzymatic reaction . A comparison of mutant enzymes with native PPases revealed that a single amino acid substitution changes the position of the mutated residue as well as the positions of several functional groups and some parts of a polypeptide chain . These changes are responsible for the fact that mutant PPases differ from the native ones in their catalytic properties . The sulfate binding to the mutant PPase active site causes molecular asymmetry, as shown for the native PPase earlier . The subunit asymmetry is manifested in different positions of sulfate and several functional groups, as well as changes in packing of hexamers in crystals and in cell parameters.

Curr Microbiol, 1998 Aug, 37(2), 108 - 16
A DNA fragment from the cyanobacterium Synechocystis sp . PCC 6803 mediates gene expression inducible by osmotic stress in E . coli; Milkowski C et al.; Fragments of Synechocystis-DNA driving salt-induced gene expression in E . coli were isolated with translational fusions to a 'lacZ gene . One fragment (fragment 19) showed a NaCl-dependent activation of betaGal expression with the maximum of a ninefold increase in enzyme activity . A similar induction was triggered by the nonionic osmolyte sucrose, indicating an osmotically dependent activation . On the contrary, transcriptional activity of the DNA fragment 19 was only slightly enhanced under salt stress conditions, suggesting a posttranscriptional mechanism of induction . Primer extension assay was performed to identify the transcription initiation site . Upstream regions share weak homology to the "-10" hexamer consensus of E . coli sigma70 promoters . The most thermodynamically stable secondary structure for the nontranslated part of the mRNA indicated that potential translation initiation sites might be blocked, leading to a low basal translation, whereas osmotic stress-induced changes of mRNA structure could be involved to increase translation . In order to analyze the function of fragment 19 in Synechocystis, promoter-probe plasmids were constructed allowing the stable integration of transcriptional and translational reporter gene fusions into the cyanobacterial chromosome . Quantitative assessment of reporter gene expression revealed a weak constitutive promoter activity of fragment 19 in Synechocystis . Sequence analysis showed that fragment 19 comprises 223 bp of the ORF sll0747 of the Synechocystis genome.

FEBS Lett, 1998 Jun 16, 429(3), 417 - 20
Mutational analysis of Glu272 in elongation factor 1A of E . coli; Mansilla F et al.; In our previous work (Mansilla et al . (1997) Protein Eng . 10, 927-934) we showed that Arg7 of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding . Substitution of Arg7 by Ala or Glu lost this activity . We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu272 (Asp284 in Thermus aquaticus) thereby binding the N-terminal region of the protein to domain 2 and thus completing the conformational rearrangement needed for binding aa-tRNA . In this work we have mutated Glu272 to arginine, either alone (Glu272Arg), or in combination with one of the above mentioned mutations (Arg7Glu/Glu272Arg) in order to test this hypothesis . Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu272 (Asp284) decreases the ability of EF1A:GTP to bind aa-tRNA.

Mol Cell, 1998 Feb, 1(3), 381 - 7
Cell cycle-dependent duplication and bidirectional migration of SeqA-associated DNA-protein complexes in E . coli; Hiraga S et al.; Using immunofluorescence microscopy, we have found that SeqA protein, a regulator of replication initiation, is localized as discrete fluorescent foci in E . coli wild-type cells . Surprisingly, SeqA foci were observed also in an oriC deletion mutant . Statistical analysis revealed that a SeqA focus is localized at midcell in newborn cells . The SeqA focus is duplicated and tethered at midcell until an FtsZ ring is formed . Subsequently, these foci migrate in opposite directions toward cell quarter sites and remain tethered there until the cell divides . The cell cycle-dependent bidirectional migration of SeqA-DNA complexes is quite different from the migration pattern of oriC Dna copies . MukB protein is required for correct localization of SeqA complexes by an unknown mechanism.

Mol Cell, 1997 Dec, 1(1), 79 - 87
Domain interactions in E . coli SRP: stabilization of M domain by RNA is required for effective signal sequence modulation of NG domain; Zheng N et al.; The E . coli protein, Fth, binds to 4.5S RNA through its M domain to form the signal recognition particle (SRP) . The other domain of Fth (NG) is a GTPase, which binds and is coordinately regulated by its receptor, FtsY . We find that the helical M domain is inherently flexible . Binding of 4.5S RNA to Fth stabilizes the M domain yet has little apparent effect on the binding of signal peptides . However, in the absence of the RNA, signal peptide binding results in a global destabilization of Fth, which is prevented by binding of 4.5S RNA . Signal peptide binding to isolated NG domain also causes a pronounced destabilization, implicating the NG domain in direct recognition of signal peptide.

Biochim Biophys Acta, 1998 May 19, 1384(2), 209 - 22
E . coli HPII catalase interaction with high spin ligands: formate and fluoride as active site probes; Maj M et al.; E . coli catalase (HPII) wild type and mutant enzymes (heme dcis-containing) were examined (i) to study the role of a distal haem cavity residue, asparagine-201, in high spin ligand binding and (ii) to compare the differences in this binding between heme d and protoheme enzymes such as that from beef liver (BLC) . High spin fluoride complexes were formed by all three HPII catalases examined, wild type (201 asn) and 201gln and 201asp mutants, but with a lower fluoride affinity than that of BLC . The binding of fluoride was pH-dependent, indicating that a proton is bound as well as a fluoride anion . HPII 201glu and 201 asp mutants showed lower affinities for fluoride than did wild type, unlike their reactions with cyanide which are essentially independent of the nature of residue 201 . The equilibria and rates of fluoride and formate binding to BLC were reexamined . The rates of reaction with formate were similar to those reported previously . Dissociation rates for fluoride-catalase are higher than for formate suggesting that the latter may be bound differently . High spin complexes between formate and all three HPII forms showed a substantially higher affinity than that of BLC for HPII wild type and progressively lower affinities for the two mutants . As with fluoride the reactions were pH-dependent, indicating that a proton is bound together with the formate anion (or that undissociated formic acid is the ligand) . The known structures of the heme groups and heme pockets involved are discussed . Formate may be bound by secondary H-bounds within the heme pocket in both heme dcis and protoheme enzymes . The nature of the heme pocket and the heme access channel may be more important than the chemical nature of the prosthetic group in controlling both high spin ligand interactions and reactions with the substrate hydrogen peroxide.

Mund Kiefer Gesichtschir, 1998 May, 2 Suppl 1, S149 - 52
{Biological activity of E . coli expressed BMP-4}; Kubler NR et al.; BMP-4 is physiologically present in low concentrations in human bone matrix . So far the protein has only been produced in small quantities by expression in mammalian cell cultures . In this study we investigated the biological activity of E . coli-expressed BMP-4 . In vitro neonatal rat muscle tissue was incubated together with BMP-4 during 4 h, followed by an incubation period of 14 days on cellulose acetate membranes in BMP-free medium . The addition of 0.4 microgram BMP-4 induced cartilage formation in 1/8 samples while 4 micrograms BMP-4 showed chondroneogenesis in 2/10 samples . When the BMP-4 concentration was increased to 40 micrograms, new cartilage formation was seen in 5/7 samples . In vivo BMP-4 was implanted intramuscularly for 3 weeks in ICR mice . Amounts of 10 micrograms rhBMP-4 and more (up to 100 micrograms) constantly induced heterotopic ossicle formation . BMP-4 was also combined with a collagen carrier and implanted for 2 and 4 weeks in the abdominal muscle of SD rats . While 0.4 microgram BMP-4 showed no bone or cartilage formation, the amount of 40 micrograms BMP-4 showed new heterotopic cartilage formation, followed by endochondral ossification in almost all samples . The results prove that E . coli-expressed BMP-4 possesses the same inductive properties as mammalian-cell-expressed BMP-4.

J Mol Biol, 1998 Jul 3, 280(1), 153 - 66
Crystal structure of the ternary complex of E . coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (Sulphate) at 2.1 A resolution; Koellner G et al.; The ternary complex of purine nucleoside phosphorylase from E . coli with formycin B and a sulphate or phosphate ion crystallized in the hexagonal space group P6122 with unit cell dimensions a=123.11, c=241.22 A and three monomers per asymmetric unit . The biologically active hexamer is formed through 2-fold crystallographic symmetry, constituting a trimer of dimers . High-resolution X-ray diffraction data were collected using synchrotron radiation (Daresbury, England) . The crystal structure was determined by molecular replacement and refined at 2.1 A resolution to an R-value of 0.196.There is one active centre per monomer, composed of residues belonging to two subunits of one dimer . The phosphate binding site is strongly positively charged and consists of three arginine residues (Arg24, Arg87 and Arg43 from a neighbouring subunit), Ser90 and Gly20 . It is occupied by a sulphate or phosphate anion, each oxygen atom of which accepts at least two hydrogen bonds or salt-bridges . The sulphate or phosphate anion is also in direct contact with the ribose moiety of formycin B . The ribose binding site is composed of Ser90, Met180, Glu181 and His4, the latter belonging to the neighbouring subunit . The base binding site is exposed to solvent, and the base is unspecifically bound through a chain of water molecules and aromatic-aromatic interactions . In all monomers the nucleosides are in the high syn conformation about the glycosidic bonds with chi in the range 100 to 130 degrees . The architecture of the active centre is in line with the known broad specificity and the kinetic properties of E . coli PNP .

Int J Radiat Oncol Biol Phys, 1998 Jul 1, 41(4), 883 - 7
Selective in vivo radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells transduced with the E . coli cytosine deaminase (CD) gene; Gabel M et al.; PURPOSE: The E . coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer . Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy . METHODS AND MATERIALS: WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model . After administration of 5-FC, tumors received 10-30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone . RESULTS: Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT . Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation . There was no appreciable toxicity using this new approach . In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization . CONCLUSION: The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity . This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers.

Mutat Res, 1998 Mar 30, 413(3), 219 - 25
Development of new tester strains derived from E . coli WP2uvrA for the determination of mutational specificity; Ohta T et al.; We have developed a set of multipurpose tester strains (WP3101 to WP3106) derived from E . coli WP2uvrA for the detection and classification of mutagens . Six kinds of F' plasmid (lacI, lacZ, proAB+) in strains CC101-CC106, each of which carried a different lacZ allele, were transferred to a delta(lac-pro) derivative of WP2uvrA . Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid in strains WP3101-WP3106 . In addition, the trpE65(ochre) allele in the same strains is available for Trp+ reversion assays . Using the new tester strains, we investigated the mutational specificities of various chemical mutagens . Base analog mutagens and alkylating mutagens induced specific types of base substitutions . G:C-->A:T transitions and G:C-->T:A transversions predominated in mutagenesis induced by 4-nitroquinoline 1-oxide . Only a slight increase in G:C-->T:A transversions was observed in cells treated with 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), although the potent mutagenicity of AF-2 was detected in a concurrent Trp+ reversion assay in the same strain . Sodium azide, on the other hand, was negative in the Trp+ reversion assay but specifically induced G:C-->A:T transitions . Present finding suggested that target sites for AF-2- and azide-induced lesions may largely depend on sequence context.

J Mol Biol, 1998 Jun 12, 279(3), 577 - 87
A nickel complex cleaves uridine in folded RNA structures: application to E . coli tmRNA and related engineered molecules; Hickerson RP et al.; To gain more insight about Escherichia coli tmRNA structure, NiCR, a square planar macrocyclic nickel (II) complex, was used to probe guanine N7 exposure . On the basis of this additional structural information, a refined secondary structure of the molecule is proposed . In addition to its known specificity for guanine N7, we show here that the chemical probe can also cleave at specific uridine residues . In contrast to the alkaline-labile modification of guanine, the reactivity of NiCR at these uridine residues results in direct strand scission . To better characterize the uridine cleavage sites and assess the importance of the RNA structure for the reaction to occur, smaller RNA molecules derived from one pseudoknot (PK4) of E . coli tmRNA containing two uridine cleavage sites were engineered and probed . It is shown that this pseudoknot can fold by itself in solution and that the expected uridine residues are also cleaved by the nickel complex, suggesting that only a local sequence and/or structural context is required for cleavage . In E . coli tmRNA, the five uridine cleavage sites are located in double-stranded regions . These sites contain a G-U wobble base-pair and a downstream uridine which is cleaved . Using smaller RNAs derived from one stem of PK4, systematic changes in the proposed recognition motif indicate that the G-U pair is required for cleavage . Furthermore, there is no cleavage if the G-U pair is reversed . If the recognition motif is moved within the stem, the cleavage site moves accordingly . Additionally, if the recognition motif is changed such that the G-U pair is flanked by two uridine residues, the reactivity occurs only at the 3' uridine . Radical quenching studies have indicated that sulfate radical, as in the case of guanine oxidation, is involved in uridine oxidation . Although additional studies are required to better characterize the reaction, this paper reports a novel specificity for a chemical probe which may be useful for investigating structural motifs involving G-U pairs in folded RNAs.

Int J Immunopharmacol, 1997 Sep-Oct, 19(9-10), 565 - 8
Antirheumatic E . coli extract OM-89 induces T cell responses to HSP60 and 70; Wendling U et al.; Oral administration of E . coli extract OM-89 is used in treating RA . It has been shown that immune reactivity to heat-shock proteins (hsp) is involved in immunomodulation of arthritis . We evaluated the postulated presence and immunogenicity of hsp's in OM-89 . The effects of OM-89 in experimental arthritis were analyzed . Proliferative T cell responses to bacterial hsp60 and hsp70 were found in rats immunized with OM-89 . And conversely, immunization with hsp antigens induced OM-89-specific T cell responses . Hsp70 (DnaK) was found to be a major immunogenic constituent of OM-89 . Parenteral immunization with OM-89 reduces resistance to adjuvant arthritis (AA), whereas oral administration protects against AA . Given the arthritis inhibitory effect of oral OM-89 in AA our findings suggest peripheral tolerance induced by hsp-specific regulatory T cells as a mode of action for OM-89 as an arthritis suppressive oral drug.

Biochemistry (Mosc), 1998 May, 63(5), 592 - 9
Changes in E . coli inorganic pyrophosphatase structure induced by binding of metal activators; Avaeva SM et al.; The three-dimensional structures of E . coli inorganic pyrophosphatase (PPase) and its complexes with Mn2+ in a high affinity site and with Mg2+ in high and low affinity sites determined by authors in 1994-1996 at 1.9-2.2 A resolution are compared . Metal ion binding initiates the shifts of alpha-carbon atoms and of functional groups and rearrangement of non-covalent interaction system of hexameric enzyme molecule . As a result, the apoPPase with six equal subunits turns after Mg2+ binding into the structure with three types of subunits distinguished by structure and occupance of the low affinity Mg2+ site . Induced asymmetry reflects the subunit interactions and cooperativity between Mg2+ binding sites . These molecular rearrangements are structural basis to account for special features of the enzyme behavior and to propose one of the pathways for enzymatic activity regulation of constitutive PPases in vivo.

J Mol Biol, 1998 May 22, 278(5), 999 - 1014
Calorimetric studies of E . coli SSB protein-single-stranded DNA interactions . Effects of monovalent salts on binding enthalpy; Kozlov AG et al.; Isothermal titration calorimetry (ITC) was used to examine the effects of monovalent salts (NaCl, NaBr, NaF and ChCl) on the binding enthalpy (DeltaHobs) for E . coli SSB tetramer binding to the single-stranded oligodeoxythymidylates, dT(pT)69 and dT(pT)34 over a wide range of salt concentrations from 10 mM to 2.0 M (25 degrees C, pH 8.1), and when possible, the binding free energy and entropy (DeltaG degrees obs, DeltaS degrees obs) . At low monovalent salt concentrations (<0.1 M), the total DeltaHobs for saturating all sites on the SSB tetramer with ssDNA shows little dependence on salt concentration, but is extremely large and exothermic (DeltaHobs=-150(+/-5) kcal/mol) . This is much larger than any DeltaHobs previously reported for a protein-nucleic acid interaction . However, at salt concentrations above 0.1 M, DeltaHobs is quite sensitive to NaCl and NaBr concentration, becoming less negative with increasing salt concentration (DeltaHobs=-70(+/-1)-kcal/mol in 2 M NaBr) . These salt effects on DeltaHobs were mainly a function of anion type and concentration, with the largest effects observed in NaBr, and then NaCl, with little effect of {NaF} . These large effects of salt on DeltaHobs appear to be coupled to a net release of weakly bound anions (Br- and Cl-) from the SSB protein upon DNA binding . However, at lower salt concentrations (</=0.1 M), specific cation effects on DeltaHobs also are observed . Under conditions where we can determine DeltaG degrees obs, DeltaS degrees obs, and DeltaHobs (25 degrees C, pH 8.1, 0.17 to 2 M NaBr), SSB binding to dT(pT)69 is enthalpically driven with a large unfavorable entropic contribution, both of which are dependent upon {NaBr} . These studies show that weak anion binding to a protein can result in large effects of salt concentration on DeltaHobs (as well as DeltaG degrees obs and DeltaS degrees obs) for a protein-ssDNA interaction . The possibility of such effects needs to be considered in any interpretation of the thermodynamics of this and other protein-nucleic acid interactions .

J Mol Biol, 1998 May 22, 278(5), 915 - 33
Association states of the transcription activator protein NtrC from E . coli determined by analytical ultracentrifugation; Rippe K et al.; The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of E . coli RNA polymerase complexed with the sigma54 factor (RNAP.sigma54) from the closed complex (RNAP.sigma54 bound at the promoter) to the open complex (melting of the promoter DNA) . This process involves phosphorylation of NtrC, assembly of a multimeric NtrC complex at the enhancer DNA sequence, interaction of this complex with promoter bound RNAP . sigma54 via DNA looping, and hydrolysis of ATP . We have used analytical ultracentrifugation to study the different NtrC association states and to derive hydrodynamic models for the conformation of the various NtrC species . The following results were obtained . (i) The unphosphorylated wild-type protein formed a dimer with a measured molecular weight of 102(+/-3) kDa, which compares to a calculated molecular weight of 54 kDa for a monomer (concentration range studied 2 to 8 microM NtrC monomer) . (ii) In the unphosphorylated state one NtrC dimer was bound to one binding site as determined with DNA oligonucleotide duplexes containing one or two binding sites (concentration range studied 50 to 1000 nM NtrC dimer) . (iii) The data obtained at protein concentrations that were below the concentration of binding sites indicate that binding to the DNA duplex with two binding sites occurred with essentially no cooperativity . The experiments were conducted in the absence of ATP . (iv) The phosphorylated protein formed a specific complex at the DNA duplex with the enhancer sequence (two NtrC binding sites) that consisted of four dimers (concentration range studied 100 to 1000 nM NtrC dimer) . (v) The formation of this octameric complex was highly cooperative, and the data suggest that two DNA strands could bind simultaneously to this complex . (vi) From the sedimentation data a model was derived in which the NtrC dimer adopts a V shaped structure with the DNA binding domains being located at the bottom and the two receiver domains at the top of the V . In this conformation higher order NtrC complexes can be stabilized by interaction between the phosphorylated receiver domain and the central activation domain of different NtrC dimers .

FEMS Microbiol Lett, 1998 Jun 1, 163(1), 65 - 72
Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E . coli; Prilipov A et al.; Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones . Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains . A series of E . coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity . This allowed preparation of 149 porin mutants in E . coli used in detailed explorations of the structure and function of this membrane protein to high resolution.

Nat Struct Biol, 1998 Jun, 5(6), 441 - 6
Crystal structure of E.coli RuvA with bound DNA Holliday junction at 6 A resolution; Hargreaves D et al.; Here we present the crystal structure of the Escherichia coli protein RuvA bound to a key DNA intermediate in recombination, the Holliday junction . The structure, solved by isomorphous replacement and density modification at 6 A resolution, reveals the molecular architecture at the heart of the branch migration and resolution reactions required to process Holliday intermediates into recombinant DNA molecules . It also reveals directly for the first time the structure of the Holliday junction . A single RuvA tetramer is bound to one face of a junction whose four DNA duplex arms are arranged in an open and essentially four-fold symmetric conformation . Protein-DNA contacts are mediated by two copies of a helix-hairpin-helix motif per RuvA subunit that contact the phosphate backbone in a very similar manner . The open structure of the junction stabilized by RuvA binding exposes a DNA surface that could be bound by the RuvC endonuclease to promote resolution.

Exp Eye Res, 1998 Feb, 66(2), 249 - 62
Soluble expression in E . coli of a functional interphotoreceptor retinoid-binding protein module fused to thioredoxin: correlation of vitamin A binding regions with conserved domains of C-terminal processing proteases; Baer CA et al.; The exchange of all-trans retinol and 11-cis retinal between the photoreceptors and retinal pigmented epithelium is mediated by interphotoreceptor retinoid-binding protein (IRBP) . IRBP contains binding sites for retinoids, docosahexaenoic acid and probably cell surface and matrix receptors . IRBP arose through the quadruplication of an ancient protein, represented by its carboxy-terminal module (module 4 in amphibians and mammals) . Module 4 has retinol binding activity and is composed of regions coded for by each of IRBP's four exons . Determining the function of the exons has been hampered by insoluble expression of module 4 in Escherichia coli . Here, we found that module 4 of Xenopus IRBP (X4IRBP), as well as its exon segments, can be expressed in a soluble form as thioredoxin fusion proteins . The recombinant proteins were purified by ion exchange and arsenical-based affinity chromatography . Liquid chromatography/mass spectrometry confirmed that the sequence of X4IRBP is correct . All-trans retinol binding was characterized by monitoring enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol . Retinol bound to X4IRBP at 2.20+/-0.29 sites with a KD=1.25+/-0.39 . One of the two sites was localized to Exons(2+3) and had a KD=0.26+/-0.13 micron . This site, which supported protein quenching and energy transfer, probably contains at least one of the two conserved tryptophans present in this segment . The second site was localized to Exon 4 . This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had a KD=1.94+/-0.20 micron . Exon 1 had no retinol binding activity . The location of the retinol binding regions correlated with the distribution of domains conserved between IRBPs and the newly recognized family of C-terminal processing proteases (CtpAs), proteins which bind and cleave non-polar carboxy termini .

Nat Biotechnol, 1998 Jun, 16(6), 566 - 71
Quantitative whole-genome analysis of DNA-protein interactions by in vivo methylase protection in E . coli; Tavazoie S et al.; A global methylation-based technique was used to identify, display, and quantitate the in vivo occupancy of numerous protein-binding sites within the Escherichia coli genome . The protein occupancy profiles of these sites showed variation across different growth conditions and genetic backgrounds . Of the 25 sites identified in this study, 24 occurred within 5' noncoding regions . Protein occupancy at 13 of these sites was supported by independent biochemical and genetic evidence . Most of the remaining 12 sites fell upstream of genes with no previously known function . A multivariate statistical analysis was utilized to group such uncharacterized genes with well-characterized ones, providing insights into their function based on a common pattern of transcriptional regulation.

Nat Biotechnol, 1998 Jun, 16(6), 541 - 6
Preparation and hybridization analysis of DNA/RNA from E . coli on microfabricated bioelectronic chips; Cheng J et al.; Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip . An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes . The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns . After the isolation, the bacteria were lysed by a series of high-voltage pulses . The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA . The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips . Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

Rapid Commun Mass Spectrom, 1998, 12(10), 630 - 6
Fingerprint matching of E . coli strains with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of whole cells using a modified correlation approach; Arnold RJ et al.; We have developed a mathematical algorithm to compare and distinguish matrix-assisted laser desorption/ionization (MALDI) mass spectra of whole bacteria cells . This fingerprint matching technique eliminates the subjectivity involved in visually comparing two spectra to determine whether they match and it provides a quantitative measure of spectral similarity . Using it, we have distinguished twenty five different strains of a single bacteria species, E . coli . Cells are grown in culture, samples are prepared, and MALDI-TOF mass spectra are recorded for each strain . Pairs of spectra are compared by a modified cross-correlation procedure . This modified approach increases the sensitivity of correlation analysis to small spectral differences . The technique can be fine-tuned by varying the number of intervals into which spectra are divided.

Glycobiology, 1998 Jun, 8(6), 633 - 6
High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity; Reddy A et al.; The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein . MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies . Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble . MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose . The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC . Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.

J Clin Microbiol, 1998 Jun, 36(6), 1608 - 11
Sensitivities and specificities of premier E . coli O157 and premier EHEC enzyme immunoassays for diagnosis of infection with verotxin (Shiga-like toxin)-producing Escherichia coli . The SYNSORB Pk Study investigators; Mackenzie AM et al.; This study describes the performance of two rapid enzyme immunoassays, Premier E . coli O157 and Premier EHEC (Meridian Diagnostics Inc., Cincinnati, Ohio) for the detection in stools of Escherichia coli O157 and verotoxins (Shiga-like toxins), respectively . Both tests were performed on stools from 876 children presenting to eight emergency departments with diarrhea . Standard culture, including E . coli O157:H7 isolation, was performed, and paired sera were taken for anti-O157-lipopolysaccharide antibody determination . Stools from patients enrolled in the study, and those yielding discordant results, were sent to a reference laboratory for repeat testing and further investigation, including cytotoxicity and non-O157 verotoxin-producing E . coli culture . Results were classified as field results (obtained in the eight site laboratories) and resolved results (obtained after repeat testing in the central laboratory) . The "gold standard" for sensitivity of both tests and for specificity of Premier E . coli O157 was isolation of E . coli O157:H7 or a fourfold anti-O157 antibody rise . Specimens positive by the Premier EHEC test and negative for E . coli O157 culture were examined for non-O157 verotoxin-producing E . coli . The field sensitivity of Premier E . coli O157 was 86%, that of Premier EHEC was 89%, and the specificity of Premier E . coli O157 was 98% . Ten of 13 discordant Premier E . coli O157 results were reassigned as true results after repeat testing . Ten non-O157 verotoxin-producing E . coli isolates were recovered from Premier EHEC-positive, E . coli O157 culture-negative stools . Only one specimen gave an unequivocally false-positive Premier EHEC result . Both tests are highly sensitive and are specific if correctly performed . The Premier EHEC test will be particularly valuable as a practical routine test for the detection of non-O157 verotoxin-producing E . coli.

Bioseparation, 1997, 7(1), 9 - 15
Pilot-scale extraction of PHB from recombinant E . coli by homogenization and centrifugation; Ling Y et al.; A new method of poly-beta-hydroxybutyrate (PHB) extraction from recombinant E . coli is proposed, using homogenization and centrifugation coupled with sodium hypochlorite treatment . The size of PHB granules and cell debris in homogenates was characterised as a function of the number of homogenization passes . Simulation was used to develop the PHB and cell debris fractionation system, enabling numerical examination of the effects of repeated homogenization and centrifuge-feedrate variation . The simulation provided a good prediction of experimental performance . Sodium hypochlorite treatment was necessary to optimise PHB fractionation . A PHB recovery of 80% at a purity of 96.5% was obtained with the final optimised process . Protein and DNA contained in the resultant product were negligible . The developed process holds promise for significantly reducing the recovery cost associated with PHB manufacture.

Gene Ther, 1998 Apr, 5(4), 507 - 13
Regional 'pro-drug' gene therapy: intravenous administration of an adenoviral vector expressing the E . coli cytosine deaminase gene and systemic administration of 5-fluorocytosine suppresses growth of hepatic metastasis of colon carcinoma; Topf N et al.; Direct administration of an adenoviral vector expressing the cytosine deaminase gene (AdCMV.CD) to tumors of colon carcinoma cells, with concomitant systemic administration of 5-fluorocytosine (5FC), results in local production of 5-fluorouracil (5FU) and suppression of tumor growth . Based on the demonstration that in vivo adenovirus-mediated gene transfer to intrahepatic tumors is relatively inefficient compared with in vivo gene transfer to hepatocytes, we developed a 'regional' prodrug strategy using in vivo Ad-mediated CD gene transfer to normal liver, permitting hepatocytes to convert 5FC into 5FU to treat local metastasis effectively in a 'trans' fashion . To show that hepatocytes can generate and export sufficient 5FU to achieve this goal, primary rat hepatocytes were exposed to AdCMV.CD and 5FC . Evaluation of the supernatants by spectrophotometry and by HPLC demonstrated significant conversion of 5FC into 5FU . When supernatants of hepatocytes exposed to AdCMV.CD and 5FC were transferred to cultures of CT26 mouse colon carcinoma cells, the CT26 viability was reduced by 80% . To show that this regional AdCMV.CD/5FC prodrug strategy can suppress tumor growth in vivo, a model of metastatic colon carcinoma was established by injecting CT26 cells into the left lobe of the liver of syngeneic Balb/c mice . The next day, AdCMV.CD was transferred to hepatocytes by intravenous administration, and 5FC treatment was started the following day . Evaluation of tumor growth after 15 days showed marked suppression of tumor growth in AdCMV.CD- and 5FC- treated animals compared to control groups (P < 0.007) . We conclude that primary hepatocytes are capable of converting 5FC into 5FU and exporting sufficient amounts of 5FU to the local milieu to suppress the growth of liver metastases of colon carcinoma cells.

Trends Biochem Sci, 1998 May, 23(5), 190 - 4
Biophysical compensation mechanisms buffering E . coli protein-nucleic acid interactions against changing environments; Record MT Jr et al.; Escherichia coli adapts to changes in growth osmolarity of at least 100-fold by making large changes in the amounts of intracellular water and solutes, including cytoplasmic K+ . A wide range of in vitro salt, solute and biopolymer concentrations should therefore be considered 'physiological' . Paradoxically, these large, osmotically induced changes in cytoplasmic K+ concentration do not greatly affect the equilibria and kinetics of cytoplasmic protein-nucleic acid interactions . Biophysical effects resulting from changes in the amount of cytoplasmic water (such as macromolecular crowding) and in the concentrations of other cytoplasmic solutes appear to compensate for the effects of changes in cytoplasmic K+ concentration and thereby maintain protein-nucleic acid equilibria and kinetics in the range required for in vivo function.

Bioorg Khim, 1998 Apr, 24(4), 293 - 9
{In vitro coupling of ATP hydrolysis to proteolysis of ATP site mutant forms of Lon-proteinase from E.coli}; Mel'nikov EE et al.; In order to identify amino acid residues involved in ATP hydrolysis by Escherichia coli protease Lon or participating in the signal transduction from the ATPase domain to the proteolytic one, potentially important residues of the ATPase domain were substituted using site-directed mutagenesis, and the properties of the resulting mutant enzymes were studied . It was found that residues K362, T363 (Walker's motif A), and D423 (motif B) are involved in the catalysis of ATP hydrolysis . K362 and T363 also participate in the system of domain-domain coupling, whereas D423 does not play a significant role in this process . Residue D387 is important for ATPase activity; however, it is not a catalytically active residue, as was earlier postulated in the literature . Residue Y493 is also involved in the signal transduction from the ATPase domain to the proteolytic one.

Thromb Haemost, 1998 May, 79(5), 1048 - 53
Attenuation of tissue thrombosis and hemorrhage by ala-TFPI does not account for its protection against E . coli--a comparative study of treated and untreated non-surviving baboons challenged with LD100 E . coli; Randolph MM et al.; This study was designed to determine the effect of a delayed infusion (T+120 min) of alanyl tissue factor pathway inhibitor (ala-TFPI) on the response to LD100 E . coli . We hypothesized that baboons treated with a low dose of TFPI (5 mg/kg) which did not survive would exhibit thrombosis, infarction and hemorrhage of target tissues such as that seen in untreated animals infused with LD100 E . coli . Eight baboons were infused with 5 mg/kg of ala-TFPI over a 10 h period beginning immediately after a 2 h infusion of LD100 E . coli (experimental group) . Four baboons were infused with E . coli followed by a 10 h infusion of saline (control group) . Of the 12 baboons, the 11 non-survivors (TFPI = 7 out of 8; controls = 4 out of 4) were evaluated for the extent of thrombosis, necrosis, hemorrhage, and congestion of target tissues and for changes in clinical chemical parameters . We expected that failure to protect would correlate with failure to inhibit thrombosis of target tissue (8) . Surprisingly ala-TFPI significantly inhibited thrombosis, hemorrhage and necrosis of adrenal and renal tissues and attenuated the rise in creatinine in the 7 treated non-survivors . The lungs of these non-survivors, however, exhibited intra-alveolar fibrin and a mild degree of hemorrhage and edema . We concluded that low doses of ala-TFPI begun as late as T+120 in minutes failed to protect against the lethal effects of LD100 E . coli in spite of completely preventing thrombosis and hemorrhage in target organs, and that thrombosis, infarction and hemorrhage of adrenal and renal tissue are not part of the lethal chain of events in this IV model of E . coli sepsis.

Vaccine, 1998 Jan, 16(1), 33 - 7
Protection against Helicobacter pylori infection in mice by intragastric vaccination with H . pylori antigens is achieved using a non-toxic mutant of E . coli heat-labile enterotoxin (LT) as adjuvant; Marchetti M et al.; We have previously shown that infection of mice with H . pylori can be prevented by oral immunization with H . pylori antigens given together with E . coli heat-labile enterotoxin (LT) as adjuvant . Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants . Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H . pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant . Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H . pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen . These results show that: (1) protection against H . pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H . pylori vaccine formulations for eventual use in humans.

Infect Immun, 1998 Jun, 66(6), 2553 - 61
Evolution of enterohemorrhagic Escherichia coli hemolysin plasmids and the locus for enterocyte effacement in shiga toxin-producing E . coli; Boerlin P et al.; This study assessed the diversity of the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene (ehxA) in a variety of Shiga toxin-producing E . coli (STEC) serotypes and the relationship between ehxA types and virulence markers on the locus for enterocyte effacement (LEE) . Restriction fragment length polymorphism of the ehxA gene and flanking sequences and of the E . coli attaching and effacing (eae) gene was determined for 79 EHEC hemolysin-positive STEC isolates of 37 serotypes . Two main groups of EHEC hemolysin sequences and associated plasmids, which corresponded to the eae-positive and the eae-negative isolates, were delineated . Comparisons of the ehxA gene sequences of representative isolates of each group showed that this gene and the rest of the EHEC hemolysin operon are highly conserved . Digestion of an ehxA PCR product with the restriction endonuclease TaqI showed a unique restriction pattern for eae-negative isolates and another one for isolates of serotypes O157:H7 and O157:NM . A conserved fragment of 5.6 kb with four potential open reading frames was identified on the EHEC hemolysin plasmid of eae-positive STEC . Phylogenetic analysis of a subset of 27 STEC isolates, one enteropathogenic E . coli isolate, and a K-12 reference isolate showed that eae-positive STEC isolates all belong to a single evolutionary lineage and that the EHEC hemolysin plasmid and the ehxA gene evolved within this lineage without recent horizontal transfer . However, the eae gene and the LEE appear to have been transferred horizontally within this STEC lineage on several occasions . The reasons for the lack of transfer or maintenance of the LEE in other STEC lineages are not clear and require further study.

J Mol Biol, 1998 Apr 24, 278(1), 147 - 65
How E . coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides; Astatke M et al.; Deoxy- and dideoxynucleotides differ only in whether they have a hydroxyl substituent at C-3' of the ribose moiety, and yet the Klenow fragment DNA polymerase prefers the natural (dNTP) substrate by several thousandfold . We have used this preference in order to investigate how Klenow fragment interacts with the sugar portion of an incoming dNTP . We screened mutant derivatives of Klenow fragment so as to identify those amino acid residues that play important roles in distinguishing between dNTPs and ddNTPs . Substitution of Phe762 with Ala or Tyr caused a dramatic decrease in the discrimination against ddNTPs, while mutations in Tyr766 and Glu710 had a smaller effect, suggesting that these two side-chains play secondary roles in the selection of dNTPs over ddNTPs.In order to understand the interactions in the enzyme-DNA-dNTP ternary complex, pre-steady-state kinetic parameters for the incorporation of dNTPs and ddNTPs were determined for wild-type Klenow fragment and for mutant derivatives that showed changes in dNTP/ddNTP discrimination . From elemental effect measurements we infer that selection against dideoxynucleotides takes place in the transition state for the conformational change that precedes phosphoryl transfer . The crucial role of the Phe762 side-chain appears to be to constrain the dNTP molecule so that the 3'-OH can make an interaction with another group within the ternary complex . When Tyr is substituted at position 762, the same interactions can take place to position the dNTP, but specificity against the ddNTP is lost because the phenolic OH can compensate for the missing 3'-OH of the nucleotide . Substitution of the smaller Ala side-chain results in a loss in specificity because the dNTP is no longer appropriately constrained . Measurement of reaction rates as a function of magnesium ion concentration suggests that the interaction made with the dNTP 3'-OH may involve a metal ion and the Glu710 side-chain, the simplest scenario being that both the 3'-OH and the carboxylate of Glu710 are ligands to the same metal ion .

J Mol Biol, 1998 Apr 3, 277(3), 647 - 62
Kinetic and X-ray structural studies of three mutant E . coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102; Stec B et al.; Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol . We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine . The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction . In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined . The enzymes crystallized in a new crystal form corresponding to the space group P6322 . Each structure has phosphate at each active site and shows little departure from the wild-type model . For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A . This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment . The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile .

Biotechniques, 1998 May, 24(5), 862 - 8
E . coli-based in vitro transcription/translation: in vivo-specific synthesis rates and high yields in a batch system; Patnaik R et al.; A highly efficient Escherichia coli-based, batch in vitro protein synthesis system using circular plasmid DNA is described . Compared to a presently available commercial kit, this improved system produced several hundredfold greater yields of the rDNA human protein thrombopoietin (ca . 450 micrograms/mL) . The system is capable of obtaining specific synthesis rates similar to those in vivo, approximately a 1000-fold increase compared to the original methods previously described . It compares favorably in rates and yields to the recently published semicontinuous methods but with the convenience of a true batch system.

Lik Sprava, 1997 Nov-Dec, (6), 63 - 6
{Antibodies to E . coli and circulating immune complexes in the bile and blood serum of patients with suppurative cholangitis}; Giulling EV et al.; It has been found out for the first time that in 55% of cases presenting with purulent cholangitis, there are antibodies to a clinical strain of E . coli in the bile taken intraoperatively from major bile duct whereas museum specimens of E . coli strain are free from the above antibodies . Immune complexes were identified in the bile of all examined patients (0.316 +/- 0.085 arbitrary units) . In the blood serum of patients with purulent cholangitis, antibodies to the clinical strain E . coli are not detectable, while there are great counts of circulating immune complexes (0.928 +/- 0.242 arbitrary units) . Based on the findings obtained a conclusion was reached about relative independence of local immunity of the biliary tract.

J Protein Chem, 1998 Apr, 17(3), 229 - 35
Catalytically active monomers of E . coli glyceraldehyde-3-phosphate dehydrogenase; Levashov PA et al.; Monomeric forms of E . coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane . Isolated monomers were shown to be catalytically active, exhibiting KM values close to the parameters characteristic of the tetrameric forms . Like tetramers, isolated monomers did not use NADP7 as a coenzyme.

Trends Biochem Sci, 1998 Apr, 23(4), 143 - 8
Responses of E . coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water; Record MT Jr et al.; Escherichia coli is capable of growing in environments ranging from very dilute aqueous solutions of essential nutrients to media containing molar concentrations of salts or nonelectrolyte solutes . Growth in environments with such a wide range (at least 100-fold) of osmolarities poses significant physiological challenges for cells . To meet these challenges, E . coli adjusts a wide range of cytoplasmic solution variables, including the cytoplasmic amounts both of water and of charged and uncharged solutes.

Prog Clin Biol Res, 1998, 397, 345 - 56
Interaction of lipopolysaccharide with a mammalian lyso-phosphatidate acyltransferase (LPAAT) transfected into E . coli, and effect of lisofylline on LPAAT transfected into mammalian cells; Bursten SL; 1 . Lipid A and LPS stimulate LPAAT activity (and hence unsaturated PA formation) in RMC membranes and whole cells . 2 . This correlates with cell phenotypic and membrane changes associated with small G proteins . 3 . Unsaturated PA and Lipid A have similar effects on cells when given exogenously . 4 . Human LPAAT-alpha and -beta isoforms were cloned and transfected into E . coli, demonstrating the ability to restore PA synthesis and reduce lyso-PA accumulation in plsC strains (LPAAT deficient mutants), as well as restoring growth at high temperatures . 5 . LPAAT transfection into E . coli plsC (JC201) strains results in an increase in LPS content, suggesting stimulation of LPS synthesis . 6 . LPAAT transfection into human A549 lung epithelial carcinoma and endothelial ECV304 cells results in increased cytokine mRNA transcription at baseline, and a significant increase in stimulated cytokine mRNA transcription . In addition, LPAAT transfection also results in increased cytokine release in response to IL-1 beta . 7 . LSF, which reduces rodent deaths in sepsis models, reduces unsaturated acyl incorporation into PA in monoblastic cell lines, and reduces serum FFA increase in human sepsis, also reduces unsaturated acyl incorporation into PA in ECV304 cells . LPAAT-alpha transfection increases linolenate incorporation into PA at the expense of linoleate incorporation, which is reversed by LSF . LPAAT-beta increases both linoleate and linolenate incorporation into PA, which is also reduced by LSF . We conclude that LPAAT and PA remodeling may play a role in diffuse renal toxicity in sepsis due to induction of cellular phenotype changes associated with PA induction by Lipid A and/or LPS . Two human isoforms of LPAAT have been cloned, and apparently address C18 unsaturated acyl chains somewhat selectively . LSF causes functional reduction in LPAAT activity in transfected systems . This does not yet imply a direct effect of LSF on LPAAT . LPAAT and LPS may interact in the membrane in a not-yet-understood manner.

Biotherapy, 1998, 10(3), 223 - 7
Oral administration of HSP-containing E . coli extract OM-89 has suppressive effects in autoimmunity . Regulation of autoimmune processes by modulating peripheral immunity towards hsp's?
Wendling U, Farine JC.
OM-89 (Subreum) is an E . coli extract used for oral administration in the treatment of rheumatoid arthritis . It contains bacterial heat shock proteins, namely hsp60 and hsp70, which were shown to be major immunogenic constituents of the drug . Immunity to bacterial heat-shock antigens was shown to be a means of immunomodulation of (experimental) autoimmune disease and possibly inflammation in general . This was demonstrated for mycobacterial hsp60 respectively hsp70 in autoimmune disease models for arthritis, diabetes and encephalitis . Parallel to the effects displayed by immunisation with hsp, oral administration of hsp-containing OM-89 was found to modify autoimmune disease in a number of animal models, such as for arthritis, diabetes and SLE . In rats immunisation with OM-89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of both E . coli and mycobacterial origin . Conversely, immunisation with hsp antigens could induce T cell reactivity specific for OM-89 . Given this and the autoimmune disease modulating properties of both hsp and OM-89 it is argued that OM-89 acts via the same mechanism as proposed for hsp: that peripheral tolerance is induced at the level of regulatory T cells with specificity for heat-shock proteins . This may constitute one mode of action for OM-89 as an arthritis suppressive oral drug in man.

Vet Res, 1998 Jan-Feb, 29(1), 89 - 98
Combined effect of ampicillin, colistin and dexamethasone administered intramuscularly to dairy cows on the clinico-pathological course of E . coli-endotoxin mastitis; Ziv G et al.; The effects of a single intramuscular injection of a drug product containing ampicillin, colistin and dexamethasone, as a suspension in a diester of propylene glycol of medium-chain fatty acids, on the clinico-pathological course of experimental Escherichia coli-endotoxin mastitis was examined in 30 dairy cows . Cows were divided into five groups, six cows per group, and 24 of them were infused with E . coli endotoxin into two quarters of their udders . The drug product was injected at 25,000 IU colistin sulphate, 10.0 mg ampicillin anhydrate and 0.025 mg dexamethasone acetate.kg-1 body weight as follows: Group 2 cows, immediately post-endotoxin infusion (PEI); Group 3 cows, 2 h PEI and, Group 4 cows, 4 h PEI . Group 1 cows were not treated with the product and served as a positive (endotoxin only) control while Group 5 cows were not challenged with endotoxin and were not treated with the product . A clinical mastitis score (CMS) was developed to quantitatively assess the degree of inflammation . Blood biochemistry and hematological parameters were used to monitor the immediate effects of treatment on several conventional inflammatory markers . Milk somatic cell counts (MSCC), milk electrical conductivity and daily milk production were among the parameters used to monitor systemic and local inflammatory reactions . Administration of the drug product immediately PEI and 2 h PEI clearly nullified some of the most severe early systemic reactions to inflammation but the effect of therapy on the local inflammatory markers was not as obvious . Notewhorthy, however, were the effects of the treatment on reducing the duration of elevated quarter MSCC and the increase in the speed of return to pre-endotoxin challenge daily milk production levels.

Dev Biol Stand, 1998, 92, 123 - 6
Adjuvant effect of non-toxic mutants of E . coli heat-labile enterotoxin following intranasal, oral and intravaginal immunization; De Magistris MT et al.; Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) are known to be very effective mucosal adjuvants, but their toxicity limits their use in humans . We genetically detoxified LT by substituting single residues in the active site of the enzymatic A subunit and obtained mutant molecules that retain mucosal adjuvant activity but are devoid of toxicity . These mutant LT molecules induce mucosal and systemic responses to antigens delivered intranasally, orally and intravaginally in mice . Furthermore, mucosal immunization with these molecules confers protection against systemic challenge with tetanus toxin (TT) and mucosal challenge with Helicobacter pylori.

Mol Cell Biochem, 1998 Jan, 178(1-2), 393 - 6
Phosphopeptides derived from in vitro phosphorylated E . coli RNA polymerase bind to DNA and affect DNA transcription; Cardellini E et al.; E . Coli RNA polymerase was phosphorylated with protein kinase CKII and allowed to bind to pBR322 . After digestion of the RNA polymerase-pBR322 complex with proteinase K, the phosphopeptides that remained bound to DNA were extracted and analyzed . These phosphopeptides are able to bind again to DNA and to inhibit transcription.

EMBO J, 1998 Mar 2, 17(5), 1526 - 34
Structural basis for MutH activation in E.coli mismatch repair and relationship of MutH to restriction endonucleases; Ban C et al.; MutS, MutL and MutH are the three essential proteins for initiation of methyl-directed DNA mismatch repair to correct mistakes made during DNA replication in Escherichia coli . MutH cleaves a newly synthesized and unmethylated daughter strand 5' to the sequence d(GATC) in a hemi-methylated duplex . Activation of MutH requires the recognition of a DNA mismatch by MutS and MutL . We have crystallized MutH in two space groups and solved the structures at 1.7 and 2.3 A resolution, respectively . The active site of MutH is located at an interface between two subdomains that pivot relative to one another, as revealed by comparison of the crystal structures, and this presumably regulates the nuclease activity . The relative motion of the two subdomains in MutH correlates with the position of a protruding C-terminal helix . This helix appears to act as a molecular lever through which MutS and MutL may communicate the detection of a DNA mismatch and activate MutH . With sequence homology to Sau3AI and structural similarity to PvuII endonuclease, MutH is clearly related to these enzymes by divergent evolution, and this suggests that type II restriction endonucleases evolved from a common ancestor.

EMBO J, 1998 Mar 2, 17(5), 1183 - 91
Crystal structure of beta-ketoacyl-acyl carrier protein synthase II from E.coli reveals the molecular architecture of condensing enzymes; Huang W et al.; In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA . The crystal structure of beta-ketoacyl synthase II from Escherichia coli has been determined with the multiple isomorphous replacement method and refined at 2.4 A resolution . The subunit consists of two mixed five-stranded beta-sheets surrounded by alpha-helices . The two sheets are packed against each other in such a way that the fold can be described as consisting of five layers, alpha-beta-alpha-beta-alpha . The enzyme is a homodimer, and the subunits are related by a crystallographic 2-fold axis . The two active sites are located near the dimer interface but are approximately 25 A apart . The proposed nucleophile in the reaction, Cys163, is located at the bottom of a mainly hydrophobic pocket which is also lined with several conserved polar residues . In spite of very low overall sequence homology, the structure of beta-ketoacyl synthase is similar to that of thiolase, an enzyme involved in the beta-oxidation pathway, indicating that both enzymes might have a common ancestor.

Biochim Biophys Acta, 1998 Feb 2, 1369(1), 131 - 40
Helicity, membrane incorporation, orientation and thermal stability of the large conductance mechanosensitive ion channel from E . coli; Arkin IT et al.; In this report, we present structural studies on the large conductance mechanosensitive ion channel (MscL) from E . coli in detergent micelles and lipid vesicles . Both transmission Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes . The secondary structure of the proteins was shown to be highly resistant towards denaturation (25-95 degrees C) based on an ellipticity thermal profile . Amide H+/D+ exchange was shown to be extensive (ca . 66%), implying that two thirds of the protein are water accessible . MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientation using dichroic ratios measured by attenuated total-reflection Fourier transform infrared spectroscopy . Here, we present and discuss this initial set of structural data on this new family of ion-channel proteins.

Bioorg Khim, 1997 Nov, 23(11), 888 - 94
{Relation between the level of E . coli gene expression and the structure of translation initiation region (TIR) . III . Sites of complementary interaction of TIR with 16S rRNA}; Gurevich AI et al.; Potential sites of the complementary interaction of the translation initiation region (TIR) with 16S rRNA are revealed, and the role of these sites in the gene expression level is studied . The high expression level of a gene depends not only on the complementary interaction of TIR with 16S rRNA in sites proximal to the start codon {anti-Shine-Dalgarno (ASD) (delta G > -8 to -10 kcal/mol) and downstream box (DB)} and located at the -15 to +20 mRNA region but also on complementary interactions in distal sites of the untranslated branch of TIR (mTIR) . Among them, the UB (upsteam box) 1 site, complementarily interacting with the exposed 452-490 segment of the 440-490 loop of 16S rRNA, may be located in the -15 to -50 mTIR segment . In the -50 to -70 mTIR segment may be located UB2 and UB3 sites, which interact with the exposed segment 478-488 of the 440-490 loop and segment 520-532 of the 520-540 loop of 16S rRNA, the UB3 site being much more efficacious . The high expression level requires that the total free energy of complementary interactions of UB1, UB2, and UB3 sites with 16S rRNA exceeds -20 kcal/mol.

Virology, 1998 Mar 15, 242(2), 387 - 94
Characterization of the brome mosaic virus movement protein expressed in E . coli; Jansen KA et al.; The biochemical and functional properties of the movement protein (MP) of brome mosaic virus (BMV) were investigated . Expression and purification of the BMV MP from Escherichia coli resulted in a pure and soluble protein preparation . Sucrose gradient centrifugation revealed that BMV MP forms oligomers consisting of two or more copies but no higher order multimers even when different ionic strengths and pHs were applied . Nitro-cellulose filter binding and gel retardation studies showed that in vitro the BMV MP preferentially bound to ss nucleic acids (RNA and DNA); the affinity to ssRNA was lower compared to BMV coat protein . The binding to ss nucleic acid was cooperative and not sequence specific and the hypothetical binding site was calculated to be around three to six nucleotides per MP monomer . The nucleic acid binding properties of the BMV MP are discussed in relation to the recent finding that this protein is also able to form tubular structures in infected protoplasts.

FEMS Microbiol Lett, 1997 Nov 15, 156(2), 175 - 85
Molecular aspects of the E . coli nucleoid protein, H-NS: a central controller of gene regulatory networks; Williams RM et al.; The nucleoid-associated protein H-NS has a central role in the structuring and control of the enteric bacterial chromosome . This protein has been demonstrated to contribute to the regulation of expression for approximately thirty genes . In this article, the molecular aspects of H-NS structure and function are briefly reviewed . H-NS contains at least two independent structural domains: a C-terminal domain, involved in the DNA-protein interactions, and a N-terminal domain, likely involved in protein-protein interactions . Recent reports have revealed that H-NS is a key factor in a multi-component gene regulatory system . Factors have now been discovered which can backup or antagonise H-NS action at certain promoters . These recent findings are summarised and discussed in relationship to the role of H-NS in DNA packaging and nucleoid structure.

Artif Cells Blood Substit Immobil Biotechnol, 1998 Jan, 26(1), 35 - 51
Growth and survival of renal failure rats that received oral microencapsulated genetically engineered E . coli DH5 cells for urea removal; Prakash S et al.; In our earlier reports we have introduced a new method for urea removal in renal failure . This is based on the oral administration of polymeric artificial cells containing nonpathogenic genetically engineered E . coli DH5 cells to renal failure rats . This resulted in the lowering of systemic uremic urea level to normal range without elevation of ammonia . The present article deals with the safety study of this approach . Microscopic study follows changes in microcapsule morphology with time are described . Two parameters, the body weight of the renal failure rats and, the survival for of the renal failure rats receiving microcapsules containing genetically engineered E . coli DH5 cells are discussed in this article . Result shows that there is no significant difference between the weight profile of uremic rats receiving oral therapy and normal control rats . Also the treated group of uremic rats survived longer than the untreated uremic rats.

Scott Med J, 1997 Dec, 42(6), 166 - 71
Seasonality and other epidemiological features of haemolytic uraemic syndrome and E . coli O157 isolates in Scotland; Douglas AS et al.; The main objective was to determine whether there was seasonality in presentation of haemolytic uraemic syndrome (HUS) in children and adults, and compare this with the reporting of E . coli O157 . The data came from Scotland, examining admissions during 1980-95 and E . coli isolates 1984-95 . Seasonality was sought by fitting a sine curve to monthly or four-weekly data throughout the year . Seasonality was present for HUS and E . coli isolates in patients under 15 years of age but not in those above that age . The highest point of the sine curve was in July/August but there was a high plateau from June to September . The timing was similar to other diarrhoeal disease . This is an epidemiological study, the purpose being to clarify the seasonal features of HUS . E . coli infection is an important food hazard and a sound knowledge of the epidemiology, could lead to optimal control . The Scottish geographic distribution is illustrated.

Virus Res, 1997 Dec, 52(2), 157 - 67
A novel poxvirus gene and its human homolog are similar to an E . coli lysophospholipase; Wall EM et al.; A novel poxvirus gene has been characterized within the genome of ectromelia virus . It has significant similarity to a family of lysophospholipases suggesting that it may function in the degradation of lysophospholipids . Since these molecules are active in the stimulation of inflammation, we hypothesize that this gene may play a role in virus virulence . This gene is expressed early in the ectromelia virus replication cycle, before DNA replication . We have also characterized a human cDNA that encodes a protein which is 49.5% identical to the ectromelia virus protein . By its presence in multiple cDNA libraries, this human gene is known to be expressed in a variety of body tissues and is likely to function in the normal regulation of lysophospholipid levels . This family of proteins have conserved blocks of amino acids that are indicative of a serine-aspartic acid-histidine catalytic triad, similar to those used by true lipases and a number of esterases.

Schweiz Arch Tierheilkd, 1997, 139(11), 479 - 84
{A molecular test for the detection of E . coli F18 receptors: a breakthrough in the struggle against edema disease and post-weaning diarrhea in swine}; Vogeli P et al.; Oedema disease and post-weaning diarrhoea in swine are associated with the colonization of the intestine with toxigenic Escherichia (E.) coli bacteria of various serotypes . Colonization depends on specific binding between adhesive fimbriae and receptors on the enterocytes . The demonstration of these receptors allows the identification of susceptible and resistant pigs . Direct sequencing of the alpha (1,2) fucosyltransferase gene (FUT1) in swine being either susceptible or resistant to adhesion by F18 fimbriated E.coli revealed a mutation at basepair 307 (M307) . Analysis of the mutation in Swiss Landrace and Large White families showed close linkage with the locus controlling resistance and susceptibility to E.coli F18 adhesion (ECF18R) . The FUT1 (M307) mutation is a good marker for selection of E.coli of F18 adhesion resistant animals . The mutation is found with variable frequencies in Duroc, Hampshire and Pietrain pigs as well.

EMBO J, 1998 Jan 2, 17(1), 93 - 100
E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF; Samartzidou H et al.; We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cation-selective OmpF . Outer membrane fractions were obtained from strains expressing just one of these porin types, and the channels were reconstituted into liposomes without prior purification . We show that the orientation of the reconstituted channels is not random and is the same for both PhoE and OmpF . Like cation-selective porins, PhoE shows fast and slow gating to closed levels of various amplitudes, testifying that the channels visit multiple functional states and behave as cooperative entities . The voltage-dependence of PhoE closure is asymmetric, but strikingly, occurs at voltages of inverse polarity from those promoting closures of OmpC and OmpF . Both slow kinetics and inverse voltage-dependence are removed when 70 amino acids from the N-terminal of OmpF are introduced into the homologous region of PhoE . This novel observation regarding the voltage-dependence of the two channel types, along with published results on PhoE and OmpF mutants, allows us to propose a molecular mechanism for voltage sensing and sensor charge movements in bacterial porins . It also offers new cues on the possible physiological relevance in bacteria of this common form of channel modulation.

J Mol Recognit, 1997 Jul-Aug, 10(4), 169 - 81
Recognition of E . coli tryptophan synthase by single-chain Fv fragments: comparison of PCR-cloning variants with the parental antibodies; Bregegere F et al.; The use of a recombinant antibody fragment instead of a complete antibody, as a conformational probe for protein structure and folding studies, can be technically advantageous provided that the recombinant fragment and its parental antibody recognize the antigen through the same mechanism . Monoclonal antibodies mAb19 and mAb93 are directed against the TrpB2 subunit of Escherichia coli tryptophan synthase and they have been extensively used as conformational probes of this protein . DNA sequences coding for single-chain variable fragments (scFv) of mAb19 and mAb93 were cloned and assembled by reverse transcription of the mRNAs from hybridomas and PCR amplification . A specialized plasmid vector, pFBX, was constructed; it enabled to express the scFvs as hybrids with the maltose-binding protein (MalE) in E . coli, and to purify them by affinity chromatography on cross-linked amylose . Six independent clones were sequenced for each hybridoma . All of them had differences in their nucleotide and amino acid sequences . A competition ELISA and the BIAcore biosensor apparatus were used to compare the energetics and kinetics with which the parental antibodies and the hybrids bound TrpB2 . The antigen binding properties of the hybrids were close to those of the parental antibodies and they were only weakly affected by the differences of sequence between the clones, with one exception . The stability of one of the hybrids and its antigen binding properties were strongly modified by a change of Gln6 into Glu, introduced into its VH domain by the PCR primers . Simple models of bimolecular interaction did not fully account for the kinetic profiles obtained with the parental antibodies and the hybrids, and this complexity suggested the existence of a conformational heterogeneity in these molecules.

Gesundheitswesen, 1997 Nov, 59(11), 656 - 60
{Epidemiologic and actual importance of entero-hemorrhagic E . coli in Northern Bavaria (1996)}; Pulz M; Following an epidemic occurrence of EHEC infections (1995-1996) Bavaria enacted legislation to survey EHEC . In consequence thereof there was an extraordinary increase in examinations for this pathogen at the Landesuntersuchungsamt fur das Gesundheitswesen Nordbayern in Erlangen . Of 2712 stool specimens 2% were positive for EHEC . At first an enzyme immunoassay (EIA) was used in screening for Shiga toxin (Stx) 1 and 2 . Because this EIA revealed a lack of specificity the PCR for amplification of sequences derived from Stx genes was set as screening test . Among 53 EHEC strains isolated by colony hybridisation 25 different serotypes could be identified . A complete virulence pattern (formation of Shiga toxins and EHEC haemolysin, presence of the eaeA gene) was detected in 3 of 4 strains from HUS patients (75%), in 14 of 17 enteritis cases (82.4%) and in 17 of 32 persons with asymptomatic carriage (53.1%) . Besides the serotypes O157H- and O103H2 strains of the serotype 08 were isolated particularly frequently in certain regions of North Bavaria for limited periods . EHEC infections in North Bavaria are related to many serotypes that are mostly different from the O157 serotype . Especially in adults the clinical course is usually asymptomatic.

FEBS Lett, 1998 Jan 23, 422(1), 52 - 6
Study of complexes of a tryptophan-free mutant of E . coli trp aporepressor with tryptophan analogues using optically detected magnetic resonance (ODMR); Ozarowski A et al.; Phosphorescence and optically detected magnetic resonance (ODMR) spectra of tryptophan (W) and several of its analogues (4-, 5-, 6-methyltryptophan (MeW); 4-, 5-, 6-fluorotryptophan (FW); 5-bromotryptophan) are compared with those of complexes formed with the W-free trp aporepressor from Escherichia coli (W19,99F) . W19,99F binds W and each analogue except 4-FW with an estimated KD < or = 30 microM; triplet state spectroscopic and kinetic effects that accompany binding at the corepressor site are reported . ODMR data for the MeW isomers are presented for the first time . No binding of 7-azaW is observed, in agreement with the low affinity found by previous workers.

Biophys Chem, 1997 Oct, 68(1-3), 95 - 102
Structure and arrangement of the delta subunit in the E . coli ATP synthase (ECF1F0); Wilkens S et al.; F1F0 type ATPases are made up of two parts, an F1, which contains three catalytic sites on beta subunits, and an F0 which contains the proton channel . These two domains have been visualized in electron microscopy as linked by a narrow stalk of around 45 A in length . Biochemical studies have provided clear evidence that the gamma and epsilon subunits are components of this stalk . There is an emerging consensus that the gamma and epsilon subunits rotate relative to the alpha 3 beta 3 domain as part of the cooperativity and energy coupling within the complex . Two other subunits are required to link the F1 to F0 in the E . coli enzyme, and these are the delta and b subunits . The structure of a major part of the delta subunit (residues 1-134) has now been obtained by NMR spectroscopy . The main feature is a six alpha-helix bundle, which provides the N-terminal domain of the delta subunit . This domain interacts with the F1 core via the N-terminal part of the alpha subunit . The C-terminal domain of delta is less well defined . This part is required for binding to the F0 part by direct interaction with the b subunits . It is argued that delta and the two copies of the b subunit are components of a second stalk linking the F1 and F0 parts, which acts as a stator to allow the energy-linked rotational movements of delta and epsilon subunits.

FEBS Lett, 1998 Jan 16, 421(3), 249 - 51
Effect of point mutations at position 89 of the E . coli 5S rRNA on the assembly and activity of the large ribosomal subunit; Zvereva MI et al.; Nucleotide residue U89 in the D loop of Escherichia coli 5S rRNA is adjacent to two domains of 23S rRNA in the large ribosomal subunit {Dokudovskaya et al., RNA 2 (1996) 146-152} . 50S ribosomal subunits were reconstituted containing U89(C, G or A) mutants of 5S rRNAs and the activities of the corresponding 70S ribosomes were studied . The U89C mutant behaves similarly to the wild-type 5S rRNA . Replacement of the pyrimidine base at position U89 by more bulky purine bases impairs the incorporation of 5S rRNA into 50S subunits, whereas the particles formed showed full activities in poly(U)-dependent poly(Phe) synthesis in the presence of either U89G or U89A 5S rRNA mutants . The activity of the reconstituted particles depends on the incorporation of 5S rRNA in agreement with early observations.

Nippon Rinsho, 1998 Jan, 56(1), 161 - 6
{Neutralizing human antibody specific for human cytomegalovirus in E . coli expression system}; Ihara S et al.; We isolated neutralizing human Fab fragment specific for human cytomegalovirus (HCMV) by phage display system . Fab libraries were constructed from peripheral lymphocyte of healthy individual . In several clones reacted for HCMV-infected HEL cells, one clone, designated 13-3, stained HCMV infected cells at 96 hrs post infection . It didn't react to cells at 6 hrs post infection or infected cells in the presence of AraC, meaning that 13-3 recognized protein synthesized at late times of infection . It also neutralized HCMV, Towne strain, efficiently . This neutralizing activity was specific for HCMV and no effect for HSV-1 and 2.

FEBS Lett, 1998 Jan 2, 421(1), 83 - 8
The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E . coli dUTP pyrophosphatase; Vertessy BG et al.; The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate (alpha,beta-imido-dUTP) . Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation . The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of alpha,beta-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion . Involvement of the C-terminal arm in alpha,beta-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141 . Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and alpha,beta-imido-dUTP, a difference not observable in C-terminally truncated dUTPase . The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.

Rev Med Chil, 1997 Mar, 125(3), 291 - 7
{Temporal variation of genotypes and serotypes of enterohemorrhagic E Coli isolated from Chilean children with intestinal infections or hemolytic uremic syndrome}; Prado V et al.; BACKGROUND: Infections by enterohemorrhagic E . Coli may be asymptomatic, may cause diarrhea, sometimes bloody or a hemolytic uremic syndrome in 2 to 7% of children . These diseases appear sporadically or as outbreaks . Host and agent factors influence the appearance of hemolytic uremic syndrome . AIM: To study the distributions and prevalence of virulence genes and enterohemorrhagic E . Coli serogroups isolated from Chilean children . MATERIALS AND METHODS: Thirty six strains isolated from children with hemolytic uremic syndrome (8 obtained in 1988-1989, 15 obtained in 1990-1993 and 13 obtained in 1995-1996), 33 strains from asymptomatic children, obtained in similar periods and 30 strains from children with bloody diarrhea, obtained in 1995-1996 were studied . Virulence factors were investigated with a colony hybridization technique using probes that identify virulence genes . Serotypes were identified with commercial antisera . RESULTS: Both SLTI and SLTII genes predominated in strains obtained from children with hemolytic uremic syndrome in 1988-1989 and 1995-1996 and SLTI gene predominated in strains obtained in 1990-1993 . Similar temporal variations in virulence genes of strains obtained from asymptomatic children were observed . SLTI/SLTII pattern predominated in strains obtained from children with bloody diarrhea and the frequency of O157 serogroup was lower, compared to strains obtained from children with hemolytic uremic syndrome . CONCLUSIONS: There was a temporal variation in toxigenic genotypes of enterohemorrhagic E . Coli strains, but no association between these genotypes and the risk for hemolytic uremic syndrome was observed.

Biochim Biophys Acta, 1998 Jan 19, 1368(2), 267 - 75
Expression and characterization of a Synechocystis PCC 6803 P-type ATPase in E . coli plasma membranes; Geisler M et al.; In a previous paper, we published the sequence of a P-type ATPase gene from Synechocystis 6803 {Geisler et al . (1993) J . Mol . Biol . 234, 1284} which showed significant homologies to eukaryotic calcium ATPases . To investigate the specificity and activities of this plasma membrane-bound enzyme, we expressed the slightly modified gene in an ATPase deficient E . coli strain . The expressed ATPase showed an apparent molecular mass of about 97kDa and is localized in the E . coli plasma membranes . The introduced 6xHis tag at the N-terminus allowed the purification of the Synechocystis 6xHis-ATPase by single-step affinity chromatography using a Ni2+-nitrilotriacetic acid resin . The ATPase activity of the enzyme is inhibited by vanadate (IC50 = 119 microM), N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, and inhibitors of eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases; however, it is stimulated by thapsigargin . Formation of phosphorylated enzyme intermediates depends on calcium ions indicating that the Synechocystis P-ATPase acts as a calcium pump equivalent to eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases.

Biochim Biophys Acta, 1998 Jan 19, 1368(2), 225 - 34
Calcium-dependent conformation of E . coli alpha-haemolysin . Implications for the mechanism of membrane insertion and lysis; Bakas L et al.; Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E . coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity . The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra . However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate . In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form . Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl . Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces . Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers . All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.

Lett Appl Microbiol, 1997 Dec, 25(6), 442 - 6
Detection of Escherichia cole O157 in French food samples using an immunomagnetic separation method and the VIDAS E . coli O157; Vernozy-Rozand C et al.; Two commercially available screening methods, an automated enzyme-linked fluorescent immunoassay (VIDAS E . coli O157) and an immunomagnetic separation followed by culture onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC), were compared for detection of Escherichia coli O157 in naturally and artificially contaminated food samples . A total of 250 naturally contaminated food samples, including raw milk cheeses, poultry, raw sausages and ground beef retail samples, were examined . Four poultry, one raw sausage and one ground beef sample were found to be positive for E . coli O157 by both methods . Of the six positive samples, five were shown to contain sorbitol-positive, O157-positive, H7-negative, motile and non-verotoxin-producing E . coli.

Mutat Res, 1997 Nov, 385(2), 151 - 7
Mutation of D . radiodurans in a gene homologous to ruvB of E . coli; Kitayama S et al.; Following the digestion of chromosomal DNA of Deinococcus radiodurans with a restriction enzyme a partial genomic library was constructed using lambda phage as a vector . A phage clone whose DNA can complement the deficiency in a radiation-sensitive mutant of D . radiodurans was isolated . Following the subcloning using phasmid vector, a hybrid plasmid containing 1.2 kb inserted DNA was obtained . After the determination of nucleotide sequence, the deduced amino acid sequence showed close homology to RuvB protein of Escherichia coli; approximately 81% of the amino acids (310 residues in total) was homologous (152 were identical and 100 amino acids were similar) . The putative protein has a conserved ATP binding domain characteristic of DNA helicases . However, we could not find an SOS promoter and ORF for RuvA protein in the sequence upstream of ruvB in contrast to the E . coli homologue . The mutant was transformed with exogenous DNA at the same rate as the wild-type cells, but it was moderately sensitive to UV, gamma-rays and to interstrand cross-linking reagents.

Biochemistry, 1997 Dec 16, 36(50), 15772 - 9
Molecular mechanism of regulation of the pyruvate dehydrogenase complex from E . coli; Hennig J et al.; The pyruvate dehydrogenase multienzyme complex from E . coli shows a sigmoidal dependency of the reaction rate on the substrate concentration when product formation is followed in the presence of physiological concentrations of the cofactor thiamin diphosphate . To elucidate the molecular mechanism of this regulation, the influence of the substrate pyruvate on the coenzyme-protein interaction has been investigated using several coenzyme analogues . The observed binding constants of all coenzymatically active analogues are increased in the presence of the substrate pyruvate, whereas those of all coenzymatically inactive analogues are not altered in the presence of pyruvate . This points to an increased binding affinity of a reaction-intermediate-coenzyme complex to the protein . Since cofactor binding and dissociation at physiological concentrations of thiamin diphosphate are slow compared to the catalytic reaction, a slow transition to the active state of the enzyme occurs . After lowering the pyruvate concentration, the opposite effect, a dissociation of the thiamin diphosphate from the enzyme is observed . This slow substrate dependent enhancement of cofactor binding enables efficient regulation of the pyruvate dehydrogenase complex by its substrate pyruvate.

Chem Biol, 1997 Nov, 4(11), 859 - 66
Aldehyde and phosphinate analogs of glutathione and glutathionylspermidine: potent, selective binding inhibitors of the E . coli bifunctional glutathionylspermidine synthetase/amidase; Lin CH et al.; INTRODUCTION: The tripeptide glutathione is converted to glutathionylspermidine (Gsp) in Escherichia coli and in trypanosomatid parasites by an ATP-cleaving Gsp synthetase activity . In parasites, an additional glutathionylation forms bis-(glutathionyl)-spermidine, trypanothione, believed to be the major surveillance thiol involved in oxidant defense mechanisms in trypanosomatid parasites . In E . coli, the Gsp synthetase is part of a bifunctional enzyme opposed by the hydrolytic Gsp amidase . RESULTS: Gsp amidase and Gsp synthetase activities of the bifunctional E . coli enzyme can be separately targeted by potent, selective slow-binding inhibitors that induce time-dependent inhibition . The inhibitor gamma-Glu-Ala-Gly.CHO most probably captures Cys59 and accumulates as the tetrahedral adduct in the amidase active site . Inhibitory Gsp phosphinate analogs are phosphorylated by ATP to yield phosphinophosphate analogs in the synthetase active site . Binding of phosphinophosphate in the Gsp synthetase active site potentiates the inhibition affinity for the aldehyde at the Gsp amidase active site by two orders of magnitude . CONCLUSIONS: Time-dependent inhibition of the Gsp amidase activity by the aldehyde substrate analog supports previous work that suggests glutathionyl acyl-enzyme intermediate formation in the Gsp amidase reaction mechanism . Use of potent selective inhibitors against Gsp amidase (aldehyde) and Gsp synthetase (phosphinate) activities provides further evidence of interdomain communication in the bifunctional enzyme from E . coli.

J Physiol Pharmacol, 1997 Dec, 48(4), 655 - 63
Increased pneumotoxicity of lipopolysaccharide from E.coli in nitric oxide deficient blood-perfused rat lungs; Bartus JB et al.; Isolated lungs of Wistar rats were ventilated by air that was enriched with 5% CO2 and perfused with homologous blood in a closed circuit (9.5 +/- 1 ml/min) using Hugo Sachs apparatus type 829 . Lipopolysaccharide from E.coli (LPS, serotype 0127:B8) at a selected sub-toxic concentration of 300 micrograms/ml added to recirculating blood produced a biphasic response . Instant transient increase in pulmonary arterial and venous perfusion pressures, and a decrease in air tidal volume, and fifty minutes later slowly progressing decrease in air tidal volume without changes in pulmonary haemodynamics, were observed . Inhibition of pulmonary nitric oxide synthase by instillation of NG-nitro-L-arginine methyl ester (L-NAME) at a final concentration of 300 microM to recirculating blood dramatically changed the response to LPS . In nitric oxide deficient lungs LPS caused prompt increase in arterial and venous pressures and a fall in air tidal volume with accompanying rise in airway resistance . Within 6.3 +/- 0.5 min a fulminant pulmonary oedema developed and all functions of the lung stopped abruptly . We conclude that pulmonary nitric oxide plays a defensive role in protecting rat lungs against LPS-induced injury.

Planta, 1998 Jan, 204(1), 120 - 6
Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E . coli; Caldwell CR et al.; Screening of a cDNA library from soybean (Glycine max (L.) Merr . cv . Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs . The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids . The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules . Escherichia coli expression systems were developed using both soybean APx cDNAs . Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products.

J Photochem Photobiol B, 1997 Nov, 41(1-2), 36 - 44
Analysis of the mutations induced in the E . coli lac Z gene by a psoralen analog; Matroule JY et al.; A psoralen in which intracyclic oxygen atoms were replaced by sulfur (7H-thieno {3,2-g}-{1}-{benzothiopyran-7-one) {PSO(S-S)}) was recently synthesized and its photobiological properties were investigated . M13mp19 DNA photosensitization mediated by PSO (S-S) followed by transfection into competent E . coli gave rise to a very low phage progeny showing the high aptitude of this compound to modify DNA . In order to characterize the role of oxidative damages in the photosensitized reaction mediated by PSO(S-S), plasmid bearing the gene encoding the formamidopyrimidine DNA glycosylase (Fpg) under the control of the inducible lac Z promoter was transfected in E . coli . Overexpression of Fpg was induced by addition of isopropyl-beta-D-thio-galactopyranoside (IPTG) to the cells and monitored by western blot analysis . Fpg overexpression did not influence the rate of M13mp19 DNA photoinactivation by PSO(S-S) neither the mutation frequency measured by the expression of beta-galactosidase encoded by the lac Z gene beared by M13mp19 . Analysis of the mutation patterns recorded with or without Fpg overexpression showed that several G to T transversions due to oxidative damages were repaired by Fpg . These data show that oxidative DNA damages generated during PSO(S-S) photosensitization have only limited biological implications measured in terms of DNA photoinactivation.

J Biomol Struct Dyn, 1997 Dec, 15(3), 611 - 7
Calliper randomization: an artificial neural network based analysis of E . coli ribosome binding sites; Nair TM; An artificial neural network based approach has been used in analyzing the translation initiation region of E . coli . The approach is based on using a trained network capable of recognizing a particular region and presenting the network with randomized calliper inputs of the true sequence . The network responds with an error when the regions which have been the main source of knowledge are randomized . Analysis of the E . coli ribosome binding sites using this approach reveal that the initiation codon and the Shine/Dalgarno sequence which are known to be important for translation initiation are also important in imparting knowledge to the network . Further, selectively changing the usually occurring initiation codon AUG, to GUG, UUG and AUU, which occur less frequently, decreases the network performance in accordance with the frequency of their occurrence . This approach can be used as a general method to derive consensus.

Biochim Biophys Acta, 1997 Dec 5, 1343(2), 221 - 6
Reversible activation of a cryptic cleavage site within E . coli beta-galactosidase in beta-galactosidase fusion proteins; Viaplana E et al.; The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E . coli from two related expression vectors . One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein . Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner . In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins . In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability . These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.

FEBS Lett, 1997 Dec 15, 419(2-3), 281 - 4
E . coli translation initiation factor IF2--an extremely conserved protein . Comparative sequence analysis of the infB gene in clinical isolates of E . coli; Steffensen SA et al.; The functionally uncharacterised N-terminal of translation initiation factor IF2 has been found to be extremely variable when comparing different bacterial species . In order to study the intraspecies variability of IF2 the 2670 basepairs nucleotide sequence of the infB gene (encoding IF2) was determined in 10 clinical isolates of E . coli . The N-terminal domains (I, II and III) were completely conserved indicating a specific function of this region of IF2 . Only one polymorphic position was found in the deduced 890 amino acid sequence . This Gln/Gly490 is located within the central GTP/GDP-binding domain IV of IF2 . The results are further evidence that IF2 from E . coli has reached a highly defined level of structural and functional development.

Nature, 1998 Jan 1, 391(6662), 99 - 102
Poly(A)- and poly(U)-specific RNA 3' tail shortening by E . coli ribonuclease E; Huang H et al.; Ribonuclease (RNase) E is an extensively studied enzyme from Escherichia coli whose site-specific endoribonuclease activity on single-stranded RNA has a central role in the processing of ribosomal RNA, the degradation of messenger RNA and the control of replication of ColE1-type plasmids . Here we report a previously undetected activity of RNase E: the ability to shorten 3' poly(A)- and poly(U)-homopolymer tails on RNA molecules . This activity, which leaves a 6-nucleotide adenylate or a 1-nucleotide uridylate remnant on primary transcripts, resides in the amino-terminal region of RNase E and does not require other protein cofactors . Addition of a 3'-terminal phosphate group prevents both removal of the poly(A) tail and endonucleolytic cleavage within primary transcripts, but has no effect on the cleavage of transcripts with tails that have already been truncated . The ability of RNase E to shorten poly(A) tails, together with the effect of tail length on endonucleolytic cleavage within primary transcripts, suggests a mechanism by which RNase E may exercise overall control over RNA decay.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 109 - 16
An E . coli B mutation, rpoB5081, that prevents growth of phage T4 strains defective in host DNA degradation; Miller ES et al.; An E . coli B Tab strain, EM121, was isolated that restricts T4 denA (DNA endonuclease II) mutants at 37 degrees C and above, but is permissive for wild-type T4 at all temperatures examined . At 42 degrees C, other mutants affected in nucleic acid metabolism (T4 dexA, regA and uvsW strains) are also restricted . Genetic analysis revealed that one mutation (rpoB5081) in the RNA polymerase beta subunit gene is sufficient for restricting all denA mutants . rpoB5081, together with a second linked mutation, is also required for restricting the other T4 mutants, rpoB5081 (P806S), previously shown to increase transcription termination in E . coli K-12, causes delayed synthesis of T4 late proteins and reduced DNA synthesis in denA infections . Thus, T4 DNA synthesis and gene expression are impaired by the rpoB5081 beta subunit when degradation of host DNA is reduced . Because the restricted T4 mutants are not readily distinguished from wild-type phage under typical plating conditions, EM121 is an important host for screening and mapping T4 denA mutations.

J Appl Physiol, 1997 Nov, 83(5), 1467 - 75
rG-CSF reduces endotoxemia and improves survival during E . coli pneumonia; Freeman BD et al.; We investigated the effects of recombinant granulocyte colony-stimulating factor (rG-CSF) during canine bacterial pneumonia . Beagles with chronic tracheostomies received daily subcutaneous rG-CSF (5 micrograms/kg body wt) or placebo for 14 days, beginning 9 days before intrabronchial inoculation with E . coli . Animals received antibiotics and fluid support; a subset received humidified oxygen (fractional inspired O2 0.40) . Compared with controls, rG-CSF increased circulating neutrophil counts (57.4 vs . 11.0 x 10(3)/mm3, day 1 after infection; P = 0.0001), decreased plasma endotoxin (7.5 vs . 1.1 EU/ml at 8 h; P < 0.01) and serum tumor necrosis factor-alpha (3,402 vs . 729 pg/ml at 2 h; P = 0.01) levels, and prolonged survival (relative risk of death = 0.45, 95% confidence interval 0.21-0.97; P = 0.038) . Also, rG-CSF attenuated sepsis-associated myocardial dysfunction (P < 0.001) . rG-CSF had no effect on pulmonary function or on blood and lung bacteria counts (all P = not significant) . Other animals challenged with endotoxin (4 mg/kg i.v.) after similar treatment with rG-CSF had lower serum endotoxin levels (7.62 vs . 5.81 log EU/ml at 6 h; P < 0.01) and less cardiovascular dysfunction (P < 0.05 to < 0.002) but similar tumor necrosis factor-alpha levels (P = not significant) compared with controls . Thus prophylactic rG-CSF sufficient to increase circulating neutrophils during bacterial pneumonia may improve cardiovascular function and survival by mechanisms that in part enhance the clearance of bacterial toxins but do not improve lung function.

Aviat Space Environ Med, 1997 Dec, 68(12), 1104 - 8
Low gravity and inertial effects on the growth of E . coli and B . subtilis in semi-solid media; Kacena MA et al.; BACKGROUND: Several published experimental results have shown that cultures of suspended bacteria exhibit increased growth in the spaceflight environment . HYPOTHESIS AND METHODS: To test whether these differences were due to fluid mechanics and not cellular effects, E . coli and B . subtilis were grown on agar cultures under static, agitated, and rotated conditions in the laboratory, and under low-gravity conditions on four Space Shuttle flights . Growth experiments were terminated with glutaraldehyde, and individual cells were counted after quantitative elution from the agar . RESULTS: The spaceflight results, in conjunction with static, rotation, and agitation experiments indicate that E . coli and B . subtilis cultures on agar, unlike their suspension grown counterparts, do not experience heightened final cell concentration when the inertial environment is changed . CONCLUSIONS: This finding points to fluid dynamics and extracellular transport phenomena and not cellular dynamics as the most likely cause of previously reported increases in bacterial growth in microgravity.

Acta Virol, 1997 Jun, 41(3), 161 - 8
Molecular cloning, sequencing, functional analysis and expression in E . coli of major core protein gene (S3) of rice dwarf virus Chinese isolate; Zhang F et al.; The complete nucleotide sequence of major core protein gene (segment S3) of rice dwarf virus (RDV) Chinese isolate was determined after cDNA cloning from the viral genomic RNA . Sequence analysis showed that the cloned fragment is 3195 bp in length and contains a single open reading frame (ORF), encoding the major core protein (P3) which M(r) of 114 K . The nucleotide and deduced amino acid sequences of S3 of this isolate share significant homology (94.1% and 97%, respectively) with those of S3 of the Japanese isolate . At the amino acid level, P3 of RDV Chinese isolate shares significant homology with P3 of rice gall dwarf virus (RGDV), significant regional homology with the rotavirus penetration, and homology with spheroidin of amsacta entomopoxvirus (SPH), which is the major protein of the occlusion body, with clp-like ATP-dependent protease binding subunit and with ATP-dependent protease ATP-binding subunit . Amino acid sequence analysis also showed that P3 contains RNA-dependent RNA polymerase (RDRP) motif-like elements such as DXXXD, SGXXXXXXN, GDD and ENXXXY . These results may suggest that P3 is a multifunctional protein which plays very important roles in the virus structure formation, virus replication and penetration processes . The full length cDNA sequence of RDV S3 and a partial one which covers nt 1004-3195 were cloned into bacterial expression vector pTrcHisB for expression . The full length cDNA sequence failed to be expressed in E . coli, but the partial sequence was successfully expressed there as confirmed by the Western blot analysis . Further analysis of RDV P3 is under way.

FEBS Lett, 1997 Oct 27, 416(3), 225 - 9
Membrane association of FtsY, the E . coli SRP receptor; de Leeuw E et al.; FtsY, the Escherichia coli homologue of the eukaryotic SRP receptor (SR alpha), is located both in the cytoplasm and in the inner membrane of E . coli . Similar to SR alpha, FtsY consists of two major domains: a strongly acidic N-terminal domain (A) and a C-terminal GTP binding domain (NG) of which the crystal structure has recently been determined . The domains were expressed both in vivo and in vitro to examine their subcellular localization . The results suggest that both domains associate with the membrane but that the nature of the association differs.

Rapid Commun Mass Spectrom, 1997, 11(17), 1900 - 8
Rapid profiling of E . coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Chong BE et al.; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to rapidly detect and profile large proteins from Escherichia coli whole cell lysates in the mass range 25-500 kDa . The bacterial samples were treated with guanidine hydrochloride and Triton X-100 to disrupt and solubilize the large inner membrane proteins . A sample preparation involving a nitrocellulose polymer film, and alpha-cyano-4-hydroxycinnamic acid, sinapinic acid or caffeic acid as matrix was utilized to rapidly monitor the presence of induced and repressed protein synthesis in response to L-arabinose catabolism in E . coli cells . The results were compared to those of 1-D or 2-D gel electrophoresis.

Acta Biol Hung, 1997, 48(3), 275 - 9
A short GC-rich sequence involved in deletion formation of cloned DNA in E . coli; Sumegi A et al.; A spontaneous deletion probably caused by rec A independent recombination between short-direct repeat sequences was observed in pUC19 plasmid carrying a piece of Streptomyces genomic DNA after culturing in liquid medium . The deletion removed an unknown portion of the cloned DNA and the BamHI-EcoRI part of the multiple cloning site with an additional flanking 111 bp from the vector . At the junction a 13 bp GC-rich DNA sequence highly homologous to a known spontaneous deletion hotspot was found.

Biochim Biophys Acta, 1997 Nov 10, 1322(1), 33 - 40
Exchangeability of the b subunit of the Cl(-)-translocating ATPase of Acetabularia acetabulum with the beta subunit of E . coli F1-ATPase: construction of the chimeric beta subunits and complementation studies; Ikeda M et al.; The gene encoding the b subunit of the Cl(-)-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids) . The deduced amino acid sequence showed high similarity to the beta subunit of the F type ATPases, but was different in the N-terminal 120 amino acids . The role of the N-terminal region was investigated using an F -ATPase beta-less mutant of E . coli, JP17 . The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction . The beta subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C) . The corresponding regions of ACLB were designated as portions A' (from 1 to 106), B' (from 107 to 172) and C' (from 173 to 478) . Chimeric proteins with combinations of A-B'-C', A-B-C' and A'-B-C restored the function as the beta subunit in E . coli F0F1-complex, but those with combinations of A'-B'-C and A-B'-C had no function as the beta subunit . These findings suggested that portion B plays an important role in the assembly and function of the beta subunit in the F0F1-complex, while portion B' of ACLB exhibited inhibitory effects on assembly and function . In addition, portion A was also important for interaction of the beta subunit with the alpha subunit in E . coli F0F1-complex . These findings also suggested that the b subunit of the Cl(-)-translocating ATPase of A . acetabulum has a different function in the Cl(-)-translocating ATPase complex, although the primary structure resembled to the beta subunit of the F1-ATPase.

Biophys Chem, 1997 Sep 1, 67(1-3), 43 - 50
A study of the dielectric properties of E . coli ribosomal RNA and proteins in solution; Bonincontro A et al.; The permittivity of ribosomal proteins and ribosomal RNA (rRNA) in solution was measured in the range 100 kHz to 1 GHz at four different temperatures (5, 15, 25 and 35 degrees C) . The experimental dielectric relaxation was analysed by the Cole-Cole equation and, from the best-fit parameters, the average values of the dipole moment and molecular radius of the proteins were obtained . The activation enthalpy was calculated from an Arrhenius plot of the relaxation time . The energy involved in the dielectric polarization of free proteins has a magnitude of about one hydrogen bond . The data on RNA were analysed according to the Mandel model . This analysis allowed the calculation of the "subunit b" as defined by Mandel . This parameter is dependent on the temperature and therefore the relaxation time does not follow the Arrhenius law . Our data thus show that, in solution, the rRNA structure is thermally rather unstable and highly flexible.

J Infect Dis, 1997 Dec, 176(6), 1625 - 8
An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E . coli; Hedberg CW et al.; An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991 . Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days . Median incubation periods ranged from 50 to 56 h . A nonmotile strain of E . coli (E . coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons . This organism produced enteroaggregative E . coli heat-stable enterotoxin 1 and contained the enteropathogenic E . coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells . E . coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.

Cell, 1997 Nov 28, 91(5), 685 - 94
The MinE ring: an FtsZ-independent cell structure required for selection of the correct division site in E . coli; Raskin DM et al.; E . coli cell division is mediated by the FtsZ ring and associated factors . Selection of the correct division site requires the combined action of an inhibitor of FtsZ ring formation (MinCD) and of a topological specificity factor that somehow prevents MinCD action at the middle of the cell (MinE) . Here we show that a biologically active MinE-Gfp fusion accumulates in an annular structure near the middle of young cells . Formation of the MinE ring required MinD but was independent of MinC and continued in nondividing cells in which FtsZ function was inhibited . The results indicate that the MinE ring represents a novel cell structure, which allows FtsZ ring formation at midcell by suppressing MinCD activity at this site.

Front Biosci, 1997 Dec 15, 2, d635 - 42
Shiga toxin mode of action in E . coli O157:H7 disease; Obrig TG; Shiga toxins (Stx) are virulence factors produced by selected bacteria pathogenic for humans . These multicomponent protein complexes are among the more potent toxins known . As inhibitors of eukaryotic protein synthesis, these toxins selectively inactivate ribosomes in an enzymatic manner . Specificity of cell targeting is determined by the high-affinity binding of Stx to its receptor, a glycosphingolipid (Gb3) located in the plasma membrane or some eukaryotic cells . Elaborated by food-borne E . coli O157:H7 bacteria, isotypes of Stx (Stx1 & Stx2) are required for the ensuing vascular changes in humans, including hemorrhagic colitis and renal hemolytic uremic syndrome . Experimental therapeutic intervention of Stx-associated disease includes the Stx receptor immobilized on biologically inert particles designed for oral presentation.

Cell, 1997 Nov 14, 91(4), 511 - 20
Enteropathogenic E . coli (EPEC) transfers its receptor for intimate adherence into mammalian cells; Kenny B et al.; Enteropathogenic E . coli (EPEC) belongs to a group of bacterial pathogens that induce epithelial cell actin rearrangements resulting in pedestal formation beneath adherent bacteria . This requires the secretion of specific virulence proteins needed for signal transduction and intimate adherence . EPEC interaction induces tyrosine phosphorylation of a protein in the host membrane, Hp90, which is the receptor for the EPEC outer membrane protein, intimin . Hp90-intimin interaction is essential for intimate attachment and pedestal formation . Here, we demonstrate that Hp90 is actually a bacterial protein (Tir) . Thus, this bacterial pathogen inserts its own receptor into mammalian cell surfaces, to which it then adheres to trigger additional host signaling events and actin nucleation . It is also tyrosine-phosphorylated upon transfer into the host cell.

Pac Symp Biocomput . 1997;:441-52.
Definite-clause grammars for the analysis of cis-regulatory regions in E . coli; Thieffry D et al.; Based on an extensive collection of sigma 70 associated regulatory mechanisms, a grammatical model has been constructed that define the functional positions and combinations of sites within DNA regulatory regions . The syntactic rules and the dictionary implemented in a Prolog program were coupled to consensus matrices used as "sensors" to integrate a syntactic recognizer . A systematic comparison between the syntactic recognizer and the standard weight matrix methodology is presented using 12 regulatory proteins and the whole collection of about 130 sigma 70 DNA regulatory regions . On the average an increased sensitivity of 5 to 10 fold is obtained with this novel approach.

Pac Symp Biocomput . 1997;:210-21.
Design of hydrophobic core of E . coli malate dehydrogenase based on the side-chain packing; Kono H et al.; We have developed computational programs for the de novo design of hydrophobic cores of proteins . The first program optimizes side-chain conformations using an updated rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest . The second program selects candidates to be engineered among the sequences by estimating changes in Gibbs free energy between the folded and unfolded structure of the proteins with new sequence . Using these programs, we constructed several variants of E . coli malate dehydrogenase (eMDH) which could have increased stability at 25 degrees C, compared to the wild type enzyme . To quantitate stability change between variants and the wild type, circular dichroism spectra were measured as a function of guanidine hydrochloride concentration at 25 degrees C, pH 7.0 . This analysis showed that three variants constructed in this study were stabilized more than or equal to the wild type . This demonstrated that our programs may be powerful tools to design new proteins with high stability.

Hua Xi Yi Ke Da Xue Xue Bao, 1996 Dec, 27(4), 348 - 53
{Subclones of fragment DNA recombinant rpDJH2 of L . interrogans serovar lai strain 017 and it's expression of high level in E . coli}; Jiang N et al.; Fragment of 1.9 kb recombinant DNA of pDJH2 was linked with vectors pT7-7 and pRSETs . Then they were transformed into E . coli JM109 (DE3) respectively . Expression of subclones was achieved in E . coli JM109 (DE3) with IPTG inducement . SDS-PAGE showed that the molecular weights of products were 68kd and 23 kd respectively . The amount of production seemed to be higher than that of the outer membrane proteins of L . interrogans serovar strain 017 in nature . Immunoblotting of pDJt and pDJrB2 (both are subclones) with the specific antiserum of anti-OMP of L . interrogans serovar lai strain 017 and the experiment of initiative immuno-protection in guinea pigs showed both protein-68 kd and 23 kd might be the antigens of immuno-protection on the outer membrane of L . interrogans serovar lai strain 017.

J Lipid Res, 1997 Oct, 38(10), 2111 - 24
Guinea pig apolipoprotein C-II: expression in E . coli, functional studies of recombinant wild-type and mutated variants, and distribution on plasma lipoproteins; Andersson Y et al.; Guinea pig apolipoprotein C-II (apoC-II) lacks four amino acid residues in the amino-terminal, lipid-binding part compared to apoC-II from other mammalian species (Andersson et al . 1991 . J . Biol . Chem . 266: 4074-4080) . To explore whether this structural difference explains the low ability of guinea pig plasma to activate lipoprotein lipase in vitro, we have expressed guinea pig apoC-II in Escherichia coli and have constructed an insertion mutant with the four missing amino acid residues compared to human apoC-II . With a synthetic emulsion of long-chain triacylglycerols, both the wild-type guinea pig apoC-II and the insertion mutant stimulated lipoprotein lipase similar to human apoC-II, but with chylomicrons from an apoC-II-deficient patient, 5- to 10-fold more of both wild-type guinea pig apoC-II and the insertion mutant were needed . Studies of tryptophane fluorescence indicated a slight difference in how guinea pig apoC-II interacted with liposomes, and presumably with lipoproteins, as compared to human apoC-II . The level of apoC-II (11.5 +/- 5.4 microg/ml) was lower in guinea pig compared to human plasma, and most of guinea pig apoC-II was on HDL-like particles . These had decreased ability to donate apoC-II to lipid emulsions compared to human HDL . Some guinea pig apoC-II was associated with LDL which, as demonstrated by surface plasmon resonance, had higher affinity for lipoprotein lipase than human LDL, and inhibited rather than stimulated the lipase reaction in vitro . We conclude that while guinea pig apoC-II is fully competent to stimulate lipoprotein lipase, the sum of several different factors explains the low ability of guinea pig plasma to accomplish stimulation.

Immunotechnology, 1996 Feb, 2(1), 37 - 46
Identification of epitope recognized by an anti-c-myc monoclonal antibody that cross-reacts with E . coli sigma factor using phage display libraries; Ikegaki N et al.; BACKGROUND: During the epitope mapping of monoclonal antibodies specific for myc proteins, two E . coli proteins cross-reactive with an anti-c-myc monoclonal antibody (MYC-X-5/1) were identified . One of the proteins is approximately 90 kDa and the other is over 150 kDa in apparent molecular mass . The molecular masses of these cross-reactive proteins suggested that they may be subunits of E . coli RNA polymerase . OBJECTIVES: We have investigated whether or not the proteins cross-reactive with MYC-X-5/1 are subunits of E . coli RNA polymerase . In addition, we have attempted to determine the epitope of MYC-X-5/1 . STUDY DESIGN: The reactivity of MYC-X-5/1 antibody was tested against highly purified E . coli RNA polymerase holo-enzyme preparations and the cell lysate made from E . coli carrying a multi-copy plasmid with an insert of the rpoD gene, the structural gene for the E . coli sigma subunit . The epitope of MYC-X-5/1 was determined by use of phage display of random peptide libraries . RESULTS: On immunoblotting assays, MYC-X-5/1 reacted with the 90-kDa protein in the E . coli RNA polymerase preparations and with the 90-kDa protein over-expressed in E . coli carrying the plasmid with the rpoD insert . In addition, we have deduced the epitope of the MYC-X-5/1 antibody to be residues 235-245 of the human c-myc protein . A highly similar sequence to this was also identified in residues 62-72 of the sigma subunit of E . coli RNA polymerase . CONCLUSION: These data demonstrated that the 90-kDa protein cross-reactive with MYC-X-5/1 is the sigma subunit of E . coli RNA polymerase . Furthermore, this study shows that random peptide libraries displayed on filamentous phage are useful tools for epitope mapping and defining cross-reactivities of monoclonal antibodies.

Immunotechnology, 1996 Jun, 2(2), 127 - 43
Affinity maturation of recombinant antibodies using E . coli mutator cells; Irving RA et al.; BACKGROUND: Phage libraries can display repertoires of antibodies which are greater in number than the mammalian immune response . However, the selected antibodies often have low binding affinity to their target antigen or hapten (KD below 10(-6) M), which is characteristic of the primary immune repertoire . There is a need for procedures to mimic somatic hypermutation through antigen driven affinity maturation, thereby increasing the affinity of selected immunoglobulins . OBJECTIVE: To investigate the effectiveness of mutation and affinity selection of recombinant antibody genes with mutator E . coli cells, incorporating phage-display strategies . STUDY DESIGN: Unique human scFvs were selected from a naive Fd-phage library . These genes were mutated by propagation in mutD5 mutator E . coli cells (mutD5-FIT) which were competent for Fd (M13) based phagemid transfections and generated point mutations (transversions and transitions) in the scFv genes . Individual phage-displayed scFvs were affinity selected from the mutation library and were assayed as soluble scFvs by ELISA and BIAcore for binding to antigen . RESULTS: The in vivo mutation of phage-displayed scFvs in E . coli mutD5-FIT, combined with affinity selection against antigen, produced scFv molecules with improved binding activity . The point mutations which resulted in single amino acid substitutions frequently produced ten fold increases in apparent binding affinity . Structural comparisons revealed that these point mutations were in framework regions (adjacent to the CDRs) and within the CDRs . In one case the apparent affinity of an anti-glycophorin scFv after mutation in the VL framework region close to CDR3 increased by 10(3) . However, this increase in apparent affinity was accompanied by an increased propensity to dimerise and form aggregates . CONCLUSIONS: A strategy for the rapid affinity maturation of scFv and Fab antibody fragments has been developed which utilises mutator strains of E . coli and incorporates phage display of antibody repertoires (libraries).

Immunotechnology, 1996 Jun, 2(2), 97 - 102
Separation of E . coli expressing functional cell-wall bound antibody fragments by FACS; Fuchs P et al.; BACKGROUND: The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria . Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene . An alternative screening system is the display of antibody fragments on bacteria . A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS) . OBJECTIVES: pAP is an expression vector for the bacterial display of antibody fragments . E . coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL) . This fusion protein binds strongly to the cell wall . To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS . STUDY DESIGN AND RESULTS: Several DNA fragments coding for various scFvs were inserted into the pAP expression vector . E . coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions . We were able to select stained E . coli expressing a specific scFv from unstained E . coli expressing a non-binding scFv by FACS . The specific selection of the bacteria was demonstrated by amplifying their genes by PCR . CONCLUSIONS: Conditions are described for separating E . coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens . These studies provide a basis for screening libraries of scFv antibodies.

Biochim Biophys Acta, 1997 Oct 23, 1329(2), 237 - 44
The construction of a cysteine-less melibiose carrier from E . coli; Weissborn AC et al.; The melibiose carrier of E . coli is a cation-sugar cotransport system . This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents . In order to investigate the importance of the cysteines, we have constructed a set of four melibiose transporters each of which has one cysteine replaced with serine or valine . The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent . In addition, we constructed a melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide . The cysteine-less melibiose carrier had no significant decrease in ability to accumulate melibiose with cotransported sodium ions or protons . Thus, none of the 4 cysteines are necessary for the function of the melibiose carrier.

Gene Expr, 1997, 6(5), 275 - 86
Display of disparate transcription phenotype by the phosphorylation negative P protein mutants of vesicular stomatitis virus, Indiana serotype, expressed in E . coli and eucaryotic cells; Mathur M et al.; The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo . We have recently shown that the P protein of VSV, New Jersey serotype (PNJ), expressed in E . coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII) . In the present work, we are studying the role of phosphorylation in the activation of the P protein of Indiana serotype (PIND), which is highly nonhomologous in amino acid sequence yet structurally similar to its New Jersey counterpart . Despite the fact that E . coli-expressed PIND required phosphorylation by CKII for activation, the phosphorylation negative P protein mutants generated by altering the phosphate acceptors S and T to alanine, surprisingly, showed transcription activity similar to wild-type in vitro . Alteration of S and T residues to phenylalanine, similarly, supported substantial transcription activity (approx . 60% of wild-type), whereas substitution with arginine residue abrogated transcription (approx . 5% of wild-type) . In contrast, the same mutants, when expressed in eucaryotic cells, exhibited greatly reduced transcription activity in vitro . This disparate display of transcription phenotype by the PIND mutants expressed in bacteria and eucaryotic cells suggests that these mutants are unique in assuming different secondary structure or conformation when synthesized in two different cellular milieu . The findings that, unless phosphorylated by CKII, the bacterially expressed unphosphorylated (P0) form of PIND, as well as the phosphorylation negative mutants expressed in eucaryotic cells, demonstrates transcription negative phenotype indicate that, like PNJ, phosphorylation of PIND is essential for its activity.

Cell, 1997 Oct 31, 91(3), 335 - 45
Crystal structure of the delta' subunit of the clamp-loader complex of E . coli DNA polymerase III; Guenther B et al.; The crystal structure of the delta' subunit of the clamp-loader complex of E . coli DNA polymerase III has been determined . Three consecutive domains in the structure are arranged in a C-shaped architecture . The N-terminal domain contains a nonfunctional nucleotide binding site . The catalytic component of the clamp-loader complex is the gamma subunit, which is homologous to delta' . A sequence-structure alignment suggests that nucleotides bind to gamma at an interdomain interface within the inner surface of the "C." The alignment is extended to other clamp-loader complexes and to the RuvB family of DNA helicases, and suggests that each of these is assembled from C-shaped components that can open and close the jaws of the "C" in response to ATP binding and hydrolysis.

Zentralbl Hyg Umweltmed, 1995 Dec, 198(2), 152 - 64
Evaluation of MUG-supplemented media for the detection of E . coli in recreational water surveillance; Neidhardt S et al.; Four media containing 4-methylumbelliferyl-beta-D-glucuronide were evaluated as a non-confirmatory procedure for E . coli detection in recreational water surveillance . The media included ECD-Agar for membrane filtration and laurylsulphate-tryptose, brilliant-green-bile and lactose as broth media in a three tube most probable number procedure . From six representative water sites, samples were collected weekly over a typical summer season (17.05-27.09.1994) and processed as parallels, using each media at two different incubation temperatures (36 degrees/44 degrees C) . Results showed that incubation temperature had no impact on E . coli counts . Each media at a given temperature could be regarded as individual enrichment procedure . None of these enrichment procedures showed a constant and predictable higher sensitivity during the sampling period at all sites compared to the others tested . For parallel results, the rate of agreement, based upon EC-guideline (76/160/EWG) staging of recreational water quality, was 85% for membrane filtration and 75% for the MPN-procedure results . Marked differences could be observed in false-positive specificity showing correlation to the selective characteristics of the media . Subsequently lactose-broth at 44 degrees C performed worst with 30% non verifiable results, while ECD-agar and laurysulphate-tryptose-broth, both at 44 degrees C, had a nearly 100% confirmation rate . Thus, combining high specificity with no lack in sensitivity these two MUG-supplemented media seem to be best suited for E . coli detection in routine recreational water surveillance.

Genet Anal, 1997 Jul, 14(2), 55 - 9
Transfer of small YACs to E . coli as large circular plasmids; Frengen E et al.; We have designed a YAC circularization vector, pCIRC3, allowing enrichment of the YAC DNA by exonuclease digestion of the linear yeast chromosomes . Due to the presence of P1 replicon sequences in this vector, the circular YACs would replicate as PACs in Escherischia coli.

Mol Cells, 1997 Aug 31, 7(4), 572 - 4
Effect of silkworm hemolymph on the expression of E . coli beta-galactosidase in insect cell lines infected with recombinant baculoviruses; Woo SD et al.; The effects of silkworm hemolymph on the expression of foreign genes by recombinant baculoviruses in cell lines were studied . The expression efficiency of beta-galactosidase by recombinant virus containing the E . coli lacZ gene at various concentrations of hemolymph and FBS was determined in BmN and Sf cell lines . The addition of hemolymph to the medium containing FBS accelerated the expression of beta-galactosidase by recombinant viruses in both cells . It was more effective in BmN cells than in Sf cells . Hemolymph was most effective in enhancing virus multiplicity under conditions of 5% FBS.

Kansenshogaku Zasshi, 1997 Sep, 71(9), 924 - 7
{A study for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E . coli) O157 by the bacterial agglutination technique}; Morooka T et al.; We evaluated the usefulness of bacterial agglutination antibodies for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E . coli) O157 infections . We examined 50 serum samples from 50 control children (whiout diarrhea 31, with diarrhea 19), 24 samples from 8 diarrhea cases due to O157:H7, 37 samples from 14 cases of hemolytic uremic syndrome (HUS) for antibodies to heat-killed E . coli E32511 (O157:H.-) strain using the bacterial agglutination technique . Of the control sera all but one (x80) showed 20 > or = in the antibody . All the diarrhea patients due to O157:H7 showed a significant rise (x160-x5120) of the titers in the sera at 5-7 days on illness, after that the titers fell rapidly . Significant antibody rise (x160-x5120) was detected in twelve out of 14 HUS patients at the early stage of the illness which fell in the convalescent phase . The assay appeared to be a useful serodiagnostic technique because of its easiness and simplicity as well as because of its high sensitivity and specificity.

Biotechnol Prog, 1997 Sep-Oct, 13(5), 692 - 4
Differences in sequence-specific expression of two anti-arsonate Fabs in E . coli; Gill DS et al.; Monoclonal antibodies are potentially useful therapeutic agents and can now be produced in hosts such as bacteria . However, it has been found that bacterial expression of some antibody-combining site fragments is greatly diminished . We compared two homologous anti-arsonate antibodies, 36-65 and 36-71, to address the question of why the former but not the latter expresses well as Fab in E . coli . These antibodies are both derived from the same variable region germline genes but differ in affinity due to somatic mutations present in 36-71 . To investigate the poor expression of 36-71 Fab, we examined several factors, such as cellular toxicity, induction with isopropylthio-beta-D-galactoside, and growth of transformed bacteria at lower temperatures (30 degrees C), as well as the possibility of E . coli strain-related expression of Fab . However, none of these factors made a significant difference to Fab expression . We next localized a significant portion of the defect in Fab expression to the heavy chain by swapping the heavy and light chains from the two antibodies to construct hybrid Fabs . We used site-directed mutagenesis to engineer amino acids into the variable regions of antibody 36-71, to reproduce those found in 36-65 which is expressed well in E . coli . The defect in expression is due to residues located in the complementarity-determining regions, as mutations of heavy chain framework residues to those present in 36-65 do not enhance expression of 36-71 Fab in E . coli.

J Biotechnol, 1997 Oct 2, 58(1), 1 - 11
Factors determining more efficient large-scale release of a periplasmic enzyme from E . coli using lysozyme; Pierce JJ et al.; Large scale use of lysozyme for periplasmic release has been impeded by the cost of the pure enzyme and its subsequent presence as a contaminant in later downstream processing steps . In this paper, we discuss the use of lysozyme for pilot scale recovery of a periplasmic enzyme from E . coli . The effects of concentration of sucrose, lysozyme and cells on periplasmic enzyme release were examined . Lysozyme concentration can be reduced 5-fold from previous reports and a reduction in sucrose concentration from 20 to 15% (w/v) allows an improvement in centrifugal harvesting by reducing viscosity . High levels of release were still achieved using this technique and further improvements in yield were obtained by optimising other components of the releasing mixture . Results show that some release is still achieved in circumstances where no lysozyme use is possible . Results also indicate that a substantial proportion (up to 70%) of lysozyme remains bound to the cellular debris after its action and is removed with this material.

Neuroreport, 1997 Sep 29, 8(14), 3005 - 8
Expression of opioid-binding cell adhesion molecule (OBCAM) and neurotrimin (NTM) in E . coli and their reactivity with monoclonal anti-OBCAM antibody; Nakajima O et al.; Opioid-binding cell adhesion molecule (OBCAM), neurotrimin (NTM) and limbic system-associated membrane protein (LAMP) are homologous and are the members of the IgLON family which is a subfamily within the immunoglobulin superfamily . We cloned the cDNAs for OBCAM and NTM, prepared recombinant proteins, and examined the reactivity of the previously prepared monocolonal anti-OBCAM antibody, OBC53, with the recombinant proteins by immunoblotting . These experiments revealed that OBC53 recognizes OBCAM about 1000 times as efficiently as NTM . Moreover, the NTM and LAMP peptides which have sequences homologous to the OBCAM peptide used for the preparation of OBC53 were 150 times less reactive to OBC53 . Thus, the OBC53 antibody is a useful tool for specifically detecting OBCAM in immunochemical experiments.

Chem Biol, 1997 Sep, 4(9), 685 - 91
Characterization of an 'orthogonal' suppressor tRNA derived from E . coli tRNA2(Gln); Liu DR et al.; BACKGROUND: In an effort to expand further our ability to manipulate protein structure, we have completed the first step towards a general method that allows the site-specific incorporation of unnatural amino acids into proteins in vivo . Our approach involves the construction of an 'orthogonal' suppressor tRNA that is uniquely acylated in vivo, by an engineered aminoacyl-tRNA synthetase, with the desired unnatural amino acid . The Escherichia coli tRNA2(Gln)-glutaminyl-tRNA synthetase (GlnRS) pair provides a biochemically and structurally well-characterized starting point for developing this methodology . To generate the orthogonal tRNA, mutations were introduced into the acceptor stem, D-loop/stem, and anticodon loop of tRNA2(Gln) . We report here the characterization of the properties of the resulting tRNAs and their suitability to severe as an orthogonal suppressor . Our efforts to generate an engineered synthetase are described elsewhere . RESULTS: Mutant tRNAs were generated by runoff transcription and assayed for their ability to be aminoacylated by purified E . coli GlnRS and to suppress an amber codon in an in vitro transcription/translation reaction . One tRNA bearing eight mutations satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E . coli aminoacyl-tRNA synthetase, including GlnRS, yet functions efficiently during protein translation . Mutations in the acceptor stem and D-loop/stem, when introduced in combination, had very different effects on the properties of the resulting tRNAs compared with the effects of the individual mutations . CONCLUSIONS: Mutations at sites within tRNA2(Gln) separated by 23-31 A interact strongly with each other, often in a nonadditive fashion, to modulate both aminoacylation activities and translational efficiencies . The observed correlation between the effects of mutations at very distinct regions of the GlnRS-tRNA and possibly the ribosomal/tRNA complexes may contribute in part to the fidelity of protein biosynthesis.

Hum Gene Ther, 1997 Sep 20, 8(14), 1637 - 44
In vivo gene therapy of cancer with E . coli purine nucleoside phosphorylase; Parker WB et al.; We have developed a new strategy for the gene therapy of cancer based on the activation of purine nucleoside analogs by transduced E . coli purine nucleoside phosphorylase (PNP, E.C . 2.4.2.1) . The approach is designed to generate antimetabolites intracellularly that would be too toxic for systemic administration . To determine whether this strategy could be used to kill tumor cells without host toxicity, nude mice bearing human malignant D54MG glioma tumors expressing E . coli PNP (D54-PNP) were treated with either 6-methylpurine-2'-deoxyriboside (MeP-dR) or arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP, fludarabine, a precursor of F-araA) . Both prodrugs exhibited significant antitumor activity against established D54-PNP tumors at doses that produced no discernible systemic toxicity . Significantly, MeP-dR was curative against this slow growing solid tumor after only 3 doses . The antitumor effects showed a dose dependence on both the amount of prodrug given and the level of E . coli PNP expression within tumor xenografts . These results indicated that a strategy using E . coli PNP to create highly toxic, membrane permeant compounds that kill both replicating and nonreplicating cells is feasible in vivo, further supporting development of this cancer gene therapy approach.

Cell, 1997 Sep 19, 90(6), 1113 - 21
Chromosome and low copy plasmid segregation in E . coli: visual evidence for distinct mechanisms; Gordon GS et al.; We have investigated DNA segregation in E . coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F . Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization . oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division . In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell . Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.

FEBS Lett, 1997 Sep 15, 414(3), 492 - 6
Cloning, purification, crystallization, and preliminary X-ray diffraction analysis of cystathionine gamma-synthase from E . coli; Wahl MC et al.; The Escherichia coli metB gene has been PCR-extracted from genomic DNA and placed under the control of a tac and a T7 promoter in plasmids pCYB1 and pET22b(+), respectively, to produce overexpressing bacterial strains for the gene product, cystathionine gamma-synthase . Efficient purification procedures have been developed for a C-terminally intein-tagged version and the wild-type target protein, yielding the product in a quantity and homogeneity amenable to high-resolution single-crystal X-ray analysis . Crystals have been obtained in space group P1 with unit cell constants a=82.2 A, b=84.2 A, c=116.2 A, alpha=107.0 degrees, beta=96.3 degrees, gamma=108.0 degrees, suggesting eight monomers per asymmetric unit (V{M}=2.23 A3/Da) . Crystals diffract to beyond 2.6 A resolution and a data set complete to 2.8 A resolution has been collected using a rotating anode X-ray source . A cryogenic buffer system has been developed to allow synchrotron data collection . Patterson self rotation searches reveal the presence of two independent tetramers with local 222 symmetry in an asymmetric unit . The crystallographic results corroborate and extend previous solution studies regarding the quaternary organization of the enzyme.

EMBO J, 1997 Aug 1, 16(15), 4617 - 27
BglF, the sensor of the E . coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein; Chen Q et al.; The Escherichia coli BglF protein is a sugar permease that is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) . It catalyses transport and phosphorylation of beta-glucosides . In addition to its ability to phosphorylate its sugar substrate, BglF has the unusual ability to phosphorylate and dephosphorylate the transcriptional regulator BglG according to beta-glucoside availability . By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and thus its ability to bind RNA and antiterminate transcription of the bgl operon . BglF has two phosphorylation sites . The first site accepts a phosphoryl group from the PTS protein HPr; the phosphoryl group is then transferred to the second phosphorylation site, which can deliver it to the sugar . We provide both in vitro and in vivo evidence that the same phosphorylation site on BglF, the second one, is in charge not only of sugar phosphorylation but also of BglG phosphorylation . Possible mechanisms that ensure correct phosphoryl delivery to the right entity, sugar or protein, depending on environmental conditions, are discussed.

J Mol Biol, 1997 Sep 19, 272(2), 190 - 9
Characterization of the interaction between the restriction endonuclease McrBC from E . coli and its cofactor GTP; Pieper U et al.; McrBC, a GTP-dependent restriction enzyme from E . coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue (PumCN40-80PumC) . The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis . We show here that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation . (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP . (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min-1 . (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC . (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD .

Cell, 1997 Sep 5, 90(5), 951 - 7
Subcellular distribution of actively partitioning F plasmid during the cell division cycle in E . coli; Niki H et al.; F plasmid is partitioned with fidelity to daughter cells during cell division cycle owing to two trans-acting genes, sopA and sopB, and a cis-acting site, sopC . We visualized the subcellular distribution of mini-F-plasmid molecules by fluorescence in situ hybridization . Mini-F-plasmid molecules having the sopABC segment were localized at midcell in newborn cells . Replicated plasmid molecules migrated to cell positions 1/4 and 3/4 without coupling with cell elongation and were tethered to these positions until completion of cell division . In contrast, molecules of a mini F plasmid lacking the sopABC segment were distributed randomly in spaces not occupied by nucleoids . The sopABC system caused replicated plasmid molecules to be positioned and tethered at the cell quarter sites.

Cell, 1997 Aug 22, 90(4), 635 - 47
Major domain swiveling revealed by the crystal structures of complexes of E . coli Rep helicase bound to single-stranded DNA and ADP; Korolev S et al.; Crystal structures of binary and ternary complexes of the E . coli Rep helicase bound to single-stranded (ss) DNA or ssDNA and ADP were determined to a resolution of 3.0 A and 3.2 A, respectively . The asymmetric unit in the crystals contains two Rep monomers differing from each other by a large reorientation of one of the domains, corresponding to a swiveling of 130 degrees about a hinge region . Such domain movements are sufficiently large to suggest that these may be coupled to translocation of the Rep dimer along DNA . The ssDNA binding site involves the helicase motifs Ia, III, and V, whereas the ADP binding site involves helicase motifs I and IV . Residues in motifs II and VI may function to transduce the allosteric effects of nucleotides on DNA binding . These structures represent the first view of a DNA helicase bound to DNA.

Anal Chem, 1997 Aug 15, 69(16), 3177 - 82
Two-dimensional analysis of recombinant E . coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry; Tang W et al.; On-line combination of capillary isoelectric focusing with electrospray ionization mass spectrometry is applied for a two-dimensional analysis of Escherichia coli proteins . The proteins are focused and cathodically mobilized in a polyacrylamide coated capillary . At the end of the capillary, various protein zones are analyzed by mass spectrometry coupled through an electrospray interface . Comparisons with silver-stained two-dimensional gel electrophoresis are made with regard to mass determination, resolution, speed, and sensitivity . Direct identification of a recombinant fusion protein of glutathione S-transferase and striped bass growth hormone is achieved without any prior protein isolation procedures.

J Korean Med Sci, 1997 Aug, 12(4), 280 - 5
Reconstitution of class I MHC molecules expressed in E . coli and complexed with single antigenic peptides; Kim J et al.; The HLA-Cw3 heavy chain has been expressed at high level as insoluble protein aggregates in E . coli . The protein aggregates dissolved in strong denaturant solution were efficiently reconstituted by removal of denaturant in the presence of an HLA-Cw3 binding peptide (FAM) and beta 2m . The reconstituted HLA-Cw3/FAM protein binds specifically to a p58 natural killer cell inhibitory receptor, a natural ligand . The HLA-A2 molecule has also been reconstituted in complex with either of a peptide from myelin associated glycoprotein (MAG) or a peptide from the GAG protein of human immunodeficiency virus . The HLA-A2/MAG protein crystallized under the identical conditions as HLA-A2 purified from human lymphoblastoid cells . The reconstitution method has yielded an abundant supply of HLA molecules complexed with single antigenic peptides, and may be of general utility in reconstituting any class I MHC molecules . However, the HLA molecules could not be reconstituted either without a peptide or with an irrelevant peptide . Using this property, the reconstitution method could be used to determine whether a peptide is restricted/bound to certain class I MHC molecule.

J Biomol Struct Dyn, 1997 Aug, 15(1), 19 - 25
Role of zinc in tRNA-acceptor stem binding by glutamyl-tRNA synthetase from E.coli: a molecular modeling study; Bothra AK et al.; A model of the N-terminal half of glutamyl-tRNA synthetase from E . coli was constructed on the basis of similarity in sequence and function of Glutaminyl- and Glutamyl-tRNA synthetases . The glutaminyl-tRNA synthetase does not contain any zinc atom, but glutamyl-tRNA synthetase from E . coli contains one atom of zinc . The specific role of zinc is not yet known . In this article, molecular modeling is employed to show that the zinc atom is well outside the contact region of the acceptor stem of tRNA . The placement of a zinc atom at a significant distance from the tRNA acceptor stem indicates that the role of zinc is likely to be indirect and structural.

J Membr Biol, 1997 Jul 15, 158(2), 137 - 45
Balance of electrostatic and hydrophobic interactions in the lysis of model membranes by E . coli alpha-haemolysin; Ostolaza H et al.; The relative weight of electrostatic interactions and hydrophobic forces in the process of membrane disruption caused by E . coli alpha-haemolysin (HlyA) has been studied with a purified protein preparation and a model system consisting of large unilamellar vesicles loaded with water-soluble fluorescent probes . Vesicles were prepared in buffers of different ionic strengths, or pHs, and the net surface charge of the bilayers was also modified by addition of negatively (e.g., phosphatidylinositol) or positively (e.g., stearylamine) charged lipids . The results can be interpreted in terms of a multiple equilibrium in which alpha-haemolysin may exist: aggregated HlyA <==> monomeric HlyA <==> membrane-bound HlyA . In these equilibria both electrostatic and hydrophobic forces are significant . Electrostatic forces become substantial under certain circumstances, e.g., membrane binding when bilayer and protein have opposite electric charges . Protein adsorption to the bilayer is more sensitive to electrostatic forces than membrane disruption itself . In the latter case, the irreversible nature of protein insertion may overcome electrostatic repulsions . Also of interest is the complex effect of pH on the degree of aggregation of an amphipathic toxin like alpha-haemolysin, since pH changes are not only influencing the net protein charge but may also be inducing protein conformational transitions shown by changes in the protein intrinsic fluorescence and in its susceptibility to protease digestion, that appear to regulate the presence of hydrophobic patches at the surface of the molecule, thus modifying the ability of the toxin to either aggregate or become inserted in membranes.

FEBS Lett, 1997 Jul 14, 411(2-3), 201 - 5
Stabilising and destabilising modifications of cysteines in the E . coli outer membrane porin protein OmpC; Gokce I et al.; Three sulfhydryl labels were used to modify two mutated sites, R37C and R74C in the eyelet of the outer membrane porin OmpC . Modification of R37C with the neutral groups Aldrithiol and bimane increases thermal stability but the negatively charged iodoacetate causes a decrease in thermal stability . The effects of substitution at R74C were less significant . Bimane labelling increases the voltage sensitivity and decreases the single channel conductance at R37C asymmetrically with smaller channels being recorded at cis negative voltages . Negatively charged acetate does not affect the voltage gating.

J Mol Biol, 1997 Jul 11, 270(2), 125 - 38
Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E . coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy; Rippe K et al.; Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C) . DNA-protein complexes were deposited on mica and images were recorded in air . The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter . By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54 . Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees . Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.

Clin Chim Acta, 1997 Jul 4, 263(1), 15 - 23
Recombinant human TSH receptor expressed in E . coli; Nakai A et al.; We expressed the extracellular domain (20-408 aa, (T) of human TSH receptor (TSHR) in E . coli to detect TSHR autoantibodies (TRAb) and, moreover, we expressed the two portions (20-218 aa (5') and 217-408 aa (3')) of the extracellular domain thought to distinguish thyroid stimulating antibodies (TSAb) from blocking antibodies (TSBAb), using pGEX.3X as the expression vector . Using Western blotting analysis of the sera from patients with autoimmune thyroid disease, sera from Graves' patients and patients with idiopathic myxedema who had TSBAb reacted with the fusion protein (T), but none of the control sera reacted with it . We further evaluated whether or not the positive sear for T recognized fusion proteins (5') or (3') . The sera from Graves' patients reacted with both fusion proteins (5') and (3') . The sera from patients with idiopathic myxedema did not react with either of fusion proteins (5') or (3') . These findings suggest that these recombinant TSHR proteins could be used as antigens to detect TRAb, and differentiate TSABb from patients with idiopathic myxedema.

J Biochem (Tokyo), 1997 Jul, 122(1), 237 - 42
Initial stage of DNA-electrotransfer into E . coli cells; Kimoto H et al.; The mechanism of electrotransfer of DNA into Escherichia coli cells was investigated under conditions optimal for genetic transformation or transfection . Simple mixing in 10% polyethylene glycol 6000 did not cause binding of DNA to the recipient bacteria . When subjected to a high electric field, however, 90-98% of the input plasmid or phage DNAs were complexed with the cells . By application of the electric field, a significant amount of biotin-labeled DNA was bound onto the recipient surface, as detected by fluorescein isothiocyanate coupled avidin . When subjected to a high voltage pulse, DNA molecules were rapidly attracted toward the anode . Concurrently, the electric field induced the orientation of bacterial cells, along the field lines and their movement toward the anode . Since the bacterial movement was relatively slow, a substantial fraction of DNA molecules must strike the cathode-facing end or side of the recipient cells . Irrespective of the high efficiency of DNA transformation, the voltage pulse did not induce release of alkaline phosphate and beta-galactosidase . The electrotransferred DNA first remained sensitive to Tris-EDTA treatment, and became refractory to spheroplasting only after incubation at 37 degrees C . These results indicate that the infecting DNA is electrophoretically plugged to the outer membrane loosened by the voltage pulse.

Cancer Gene Ther, 1997 Jul-Aug, 4(4), 229 - 38
Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E . coli nitroreductase gene; Green NK et al.; Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy . CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA . We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines . A clone of LS174T cells expressing nitroreductase showed > 50-fold increased sensitivity to CB1954, and nitroreductase-expressing clones of pancreatic tumor lines were up to approximately 500-fold (SUIT2) more sensitive than parental cells . Concentrations of CB1954 minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with SUIT2 cells expressing nitroreductase; and marked "bystander" cell killing was seen with just 10% of cells expressing nitroreductase . Significant bystander cell killing was dependent on a high cell density . In conjunction with regional delivery of vectors and tumor selectivity of cell entry and/or gene expression, nitroreductase and CB1954 may be an attractive combination for prodrug-activating enzyme gene therapy of colorectal and pancreatic cancer.

EMBO J, 1997 Jul 1, 16(13), 4126 - 33
Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner; Freistroffer DV et al.; Ribosomes complexed with synthetic mRNA and peptidyl-tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis . The peptide-releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non-hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide . With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide . RF3 hydrolysis of GTP to GDP + P(i) was dependent only on ribosomes and not on RF1 or RF2 . RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release . A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.

FEMS Microbiol Lett, 1997 Jul 1, 152(1), 95 - 100
The traditional enteropathogenic Escherichia coli (EPEC) serogroup O125 comprises serotypes which are mainly associated with the category of enteroaggregative E . coli; do Valle GR et al.; Genotypic and phenotypic virulence markers of the different categories of diarrheagenic Escherichia coli were investigated in 76 strains of the enteropathogenic E . coli (EPEC) serogroup O125 . The most frequent serotype found was O125ac:H21 . None of the serotypes behaved as EPEC, i.e . carried the eaeA, bfpA, and EAF DNA sequences simultaneously and presented localized adherence to HeLa cells . All strains of O125ac:H6 were atypical EPEC since they carried eaeA only, and presented an indefinite pattern of adherence . All strains of O125ab:H9, O125ac:H9, O125?:H16, and O125ab:H21 and 79% of the O125ac:H21 strains were enteroaggregative E . coli, since they carried a specific DNA sequence and presented the typical aggregative adherence pattern.

Mol Cells, 1997 Jun 30, 7(3), 394 - 8
Monitoring catecholamine differentiation in the embryonic brain and peripheral neurons using E . coli lacZ as a reporter gene; Kim SJ et al.; An X-gal based histochemical assay was used to detect catecholamine (CA) cells in transgenic mouse embryos, in which the expression of the lacZ reporter was driven by the tissue-specific promoter of the rat tyrosine hydroxylase (TH) gene . As the first enzyme in the biosynthetic pathway for CA neurotransmitters, TH is a specific phenotypic marker for CA cells in the central and peripheral nervous systems of adult animals . During embryogenesis, TH expression appears permanently within CA-producing cells, and transiently within several other cell types . In this study we were able to monitor TH expression in transgenic mouse embryos by following the expression of the lacZ reporter in substantia nigral dopaminergic neurons in the central nervous system, the trigeminal (V) sensory ganglia, and dorsal root ganglia in the periphery . Our results demonstrate that the rat TH promoter-lacZ transgene provides an important experimental tool for monitoring catecholaminergic lineage cells during embryogenesis.

Nature, 1997 Jun 12, 387(6634), 703 - 5
Evolution of high mutation rates in experimental populations of E . coli; Sniegowski PD et al.; Most mutations are likely to be deleterious, and so the spontaneous mutation rate is generally held at a very low value . Nonetheless, evolutionary theory predicts that high mutation rates can evolve under certain circumstances . Empirical observations have previously been limited to short-term studies of the fates of mutator strains deliberately introduced into laboratory populations of Escherichia coli, and to the effects of intense selective events on mutator frequencies in E . coli . Here we report the rise of spontaneously originated mutators in populations of E . coli undergoing long-term adaptation to a new environment . Our results corroborate computer simulations of mutator evolution in adapting clonal populations, and may help to explain observations that associate high mutation rates with emerging pathogens and with certain cancers.

Gene, 1997 Jun 11, 192(1), 33 - 8
Biogenesis of the bundle-forming pilus of enteropathogenic Escherichia coli: reconstitution of fimbriae in recombinant E . coli and role of DsbA in pilin stability--a review; Donnenberg MS et al.; Enteropathogenic Escherichia coli (EPEC) adhere to tissue culture cells in a distinct pattern known as localized adherence (LA) . We have defined two loci necessary for LA . A plasmid-encoded gene cluster encodes bundlin, the major structural subunit of a type-IV fimbria called the bundle-forming pilus (BFP), a prepilin peptidase necessary for processing of pre-bundlin to its mature form, and twelve other proteins . Under the control of an exogenous promoter, these 14 genes are sufficient for the biogenesis of BFP in a heterologous E . coli host . The chromosomal gene dsbA, which encodes a periplasmic disulfide-bond oxidoreductase, is also required for LA . In the absence of DsbA protein, bundlin is made but rapidly degraded . Pre-bundlin is also rapidly degraded in the absence of DsbA, suggesting that the prepilin is a transcytoplasmic protein simultaneously accessible to enzymes on both sides of the inner membrane . These studies offer a fresh perspective on the biogenesis of type-IV pili.

Gene, 1997 Jun 3, 191(2), 167 - 72
Cloning of a rat cDNA encoding retinal dehydrogenase isozyme type I and its expression in E . coli; Penzes P et al.; Peptides sequenced from the purified rat liver cytosolic retinal dehydrogenase P1 {Posch, K.C., Burns, R.D . and Napoli, J.L., 1992 . Biosynthesis of all-trans-retinoic acid from retinal: recognition of retinal bound to cellular retinol-binding protein (type I) as substrate by a purified cytosolic dehydrogenase . J . Biol . Chem . 267, 19676-19682} were used to design oligonucleotides for cloning its cDNA . The deduced amino acid sequence of P1, now designated retinal dehydrogenase type I or RalDH(I), has close similarity with mouse AHD-2 and rat kidney aldehyde dehydrogenase, but is distinct from rat phenobarbital-inducible aldehyde dehydrogenase (PIADH), the presumed rat liver homolog of mouse AHD-2 . Rat kidney (100%) and lung (88%) show relatively high mRNA levels of RalDH(I), liver (34%) and brain (22%) have moderate levels, and testis (8%) has low levels . Retinoid status affects RalDH(I) mRNA levels differently in different tissues . E . coli-expressed RalDH(I) exhibits allosteric kinetics for retinal with a Hill coefficient of 1.7, a K0.5 value of 1.4 microM and a Vmax of 52 nmol min(-1) mg(-1) protein . These data establish the cospecificity of P1 and RalDH(I), show that retinoid status affects expression of its mRNA in a tissue-dependent manner, and illustrate that aldehyde dehydrogenase isozymes with extensive homology can participate in different metabolic paths, e.g., RalDH vs . PIADH.

Berl Munch Tierarztl Wochenschr, 1997 Jun, 110(6), 211 - 3
{Present problems in detection of sources of infection and chains of infection with enterohemorrhagic E . coli (EHEC)}; Weber A et al.; In the context of the detection of the haemolyticuraemic syndrome (HUS) and of enterohaemorrhagic E . coli (EHEC) in 13 persons, 372 faeces samples from 13 herds of cattle in northern Bavaria were examined for the presence of EHEC . 128 (34.4%) of the faeces samples were found to be VT-positive . From 78 of these samples (61%), verotoxin-producing E . coli strains (VTEC) could be isolated . During these examinations, E . coli strains with combinations of markers (VT, eae A, EHEC haemolysin) being typical of EHEC were found in 3 samples from animals belonging to the same herd . In 2 cases, these could be assigned to O157:H-, in one, to O118:H16 . It has not been possible to detect possible sources of infection and chains of infection assumed to exist in association with the detection of HUS and EHEC infections in humans because most cases had been diagnosed on the basis of verotoxin detection in stool specimens . Moreover, corresponding isolates for a comparative onward differentiation from verotoxin-producing E . coli isolates from animals were not or could not be made available.

Zentralbl Bakteriol, 1997 Jun, 286(1), 1 - 8
Cloning of the PacB-Ter region from plasmid Mip233 (IncHI3) and their expression in E . coli ton, tol mutants; Vilchez G et al.; A region of the plasmid Mip233 (incompatibility group HI3) encoding the phenotypes of resistance to the channel-forming colicins (character PacB) and potassium tellurite (Ter), was cloned and studied . Both properties are contained in an insert of 2.2 Kbp, being the smallest functional clone (pB22) isolated so far . E . coli DH5 alpha pB22 transformants exhibit resistance to the colicins as well as to high levels of tellurite (> 1000 micrograms ml-1) . Results suggest that they are genetically linked forming an inducible operon . pB22 does not show significant homology with DNA from other H plasmids . Tests using E . coli ton and tol mutants harbouring recombinant pB22 indicate that the product of gene tolC, but not that of tonB, is required for the expression of the PacB and Ter phenotypes.

J Hepatol, 1997 Jun, 26(6), 1179 - 86
Evaluation of hepatitis C virus envelope proteins expressed in E . coli and insect cells for use as tools for antibody screening; Hussy P et al.; BACKGROUND/METHODS: The two envelope proteins of hepatitis C virus, E1 and E2, were expressed in E . coli and, as secretory proteins, in Sf9 insect cells using recombinant baculoviruses . Co-infection of insect cells with E1 and E2-recombinant baculoviruses was performed, which has been shown to result in formation of E1-E2 dimers . All envelope proteins were purified by Ni2+-NTA chromatography and used for screening of serum samples in a HCV EIA assay . Serum samples of normal blood donors, chronically HCV-infected patients, a mixed titer panel and several seroconversion panels were screened and compared to test results with Cobas Core Anti-HCV EIA . RESULTS: Screening of the sera of chronically HCV-infected patients (100% positive in Cobas Core Anti-HCV EIA) revealed 10-40% anti-E1 positive sera using different Sf9-expressed, glycosylated proteins and 93% using E . coli-expressed, non-glycosylated E1 protein . When the same sera were tested with different E2 proteins expressed in Sf9 cells and in E . coli, about 70-73% showed anti-E2 reactivity . When the proteins from Sf9 cells co-infected with E1- and E2-recombinant baculoviruses were tested, 70-80% of the same sera showed anti-envelope reactivity . CONCLUSIONS: Testing of these patient antisera, and those from the well-characterized mixed titer panel BBI-PHV203, showed that recombinant E1 expressed in E . coli and co-expressed E1 and E2 proteins from Sf9 cells could be used as additional tools for anti-HCV antibody screening.

Proteins, 1997 Jun, 28(2), 285 - 8
Expression, crystallization and preliminary X-ray diffraction study of FtsY, the docking protein of the signal recognition particle of E . coli; Montoya G et al.; FtsY is the docking protein or SR alpha homologue in E . coli . It is involved in targeting secretory proteins to the cytoplasmic membrane by interacting with the signal recognition particle, controlled by guanosine 5'-triphosphate . Two different constructs have been used in crystallization studies: the full-length protein and a truncated fragment with a his-tag at the C terminus . Only the second construct resulted in crystals suitable for x-ray diffraction . The crystals belong to the monoclinic space group P2(1) with cell dimensions a = 32.20 A, b = 79.57 A, c = 59.21 A, and beta = 94.45, and contain one molecule per asymmetric unit . At cryogenic temperatures the crystals diffract to a resolution limit of 2.5 A by using a rotating anode, and beyond 1.8 A by using synchrotron radiation.

Cell, 1997 May 16, 89(4), 607 - 17
In vitro reconstitution of the late steps of genetic recombination in E . coli; Eggleston AK et al.; Purified proteins have been used to reconstitute an in vitro system for the medial-to-late stages of recombination in E . coli . In this system, RecA protein formed recombination intermediates that were processed by the actions of the RuvA, RuvB, and RuvC proteins . RuvAB was found to promote branch migration, to dissociate the RecA filament, and to modulate the orientation of cleavage of Holliday junction resolution by RuvC . Monoclonal antibodies directed against RuvA, RuvB, or RuvC inhibited resolution in the reconstituted system . Specific protein-protein interactions between the branch migration motor (RuvB) and the resolvase (RuvC) were also observed . These results provide evidence for coordinated action during the late stages of recombination, possibly involving the assembly of a RuvABC branch migration/resolution complex.

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