Bioorg Med Chem, 2005 Feb 1, 13(3), 909 - 15 A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E . coli as TEV protease cleavable fusion protein; Tolbert TJ et al.; We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins . Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines . The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively . The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly. J Microbiol Methods, 2005 Mar, 60(3), 375 - 82 Characterisation of E . coli O157 isolates from bovine hide and beef trimming in Irish abattoirs by pulsed field gel electrophoresis; Duffy G et al.; Escherichia coli O157 isolates from bovine hide (n=117) and beef trimmings (n=32) from a single abattoir were examined by pulsed field gel electrophoresis (PFGE) . Using BioNumerics software, dendrograms of isolates from each sample type (i.e . hide and beef trimming) were produced . In assessing the genetic relatedness of isolates, a similarity criterion of 80% was applied . The 117 E . coli O157 hide isolates were grouped into 14 clusters, comprising of 109 different PFGE profiles . Of the 109 different PFGE profiles, 8 were common to multiple isolates (i.e . shared 100% similarity by PFGE) . The 32 E . coli O157 beef trimming isolates produced 28 different PFGE profiles and 2 clusters . Of the 28 PFGE profiles, 2 were common to multiple isolates and the remaining 26 were distinct . On a number of sampling occasions, isolates displaying identical PFGE patterns were recovered from multiple isolates collected from a single sample type (i.e . hides or trimmings), suggesting cross contamination from contaminated hides/animals to uncontaminated hides/animals and from contaminated beef trimmings to uncontaminated beef trimmings during abattoir operations. J Mol Biol, 2005 Feb 4, 345(5), 1171 - 83 Epub 2004 Dec 16. Identification of Single Mn(2+) Binding Sites Required for Activation of the Mutant Proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray Crystallography; Tsunaka Y et al.; Escherichia coli RNase HI has two Mn(2+)-binding sites . Site 1 is formed by Asp10, Glu48, and Asp70, and site 2 is formed by Asp10 and Asp134 . Site 1 and site 2 have been proposed to be an activation site and an attenuation site, respectively . However, Glu48 and Asp134 are dispensable for Mn(2+)-dependent activity . In order to identify the Mn(2+)-binding sites of the mutant proteins at Glu48 and/or Asp134, the crystal structures of the mutant proteins E48A-RNase HI*, D134A-RNase HI*, and E48A/D134N-RNase HI* in complex with Mn(2+) were determined . In E48A-RNase HI*, Glu48 and Lys87 are replaced by Ala . In D134A-RNase HI*, Asp134 and Lys87 are replaced by Ala . In E48A/D134N-RNase HI*, Glu48 and Lys87 are replaced by Ala and Asp134 is replaced by Asn . All crystals had two or four protein molecules per asymmetric unit and at least two of which had detectable manganese ions . These structures indicated that only one manganese ion binds to the various positions around the center of the active-site pocket . These positions are different from one another, but none of them is similar to site 1 . The temperature factors of these manganese ions were considerably larger than those of the surrounding residues . These results suggest that the first manganese ion required for activation of the wild-type protein fluctuates among various positions around the center of the active-site pockets . We propose that this fluctuation is responsible for efficient hydrolysis of the substrates by the protein (metal fluctuation model) . The binding position of the first manganese ion is probably forced to shift to site 1 or site 2 upon binding of the second manganese ion. Yi Chuan, 2004 Mar, 26(2), 202 - 4 {Conserved Region Analysis of aac(3) IIGene From E.coli Aminoglycoside Resistance Strain.}; Chen JG et al.; According to standard K B method,bacteriostatic tests were performed to screen out aminoglycoside resistance bacteria from 47 strains of isolated E.coli.To analyze correlations between the degree of E.coli aminoglycoside resistance and aac(3) II gene conserved region,PCR amplified aac(3) II gene conserved regions and were analyzed by DNA sequencing.The results showed that there were two species of aac(3) IIgene type including 65G and 84T or 65A and 84C in the samples.Strains with high activity of modifying enzyme to gentamicin all were 65G and 84T aac(3) II gene type. World J Gastroenterol, 2005 Jan 21, 11(3), 454 - 6 Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli; Kang QZ et al.; AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori (H pylori) neutrophil-activating protein (NAP) and E . coli maltose-binding protein (MBP) and to evaluate its immunoreactivity and immunogenicity . METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x . The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E . coli TB1 . Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis . Soluble rMBP-NAP was purified by amylose affinity chromatography . Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment, Western blotting with human H pylori anti-sera . RESULTS: E.coli TB(1) carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG . The molecular weight of rBMP-NAP was about 57 kD, accounting for 37.55% of the total protein in the sonicated supernatant of E . coli TB(1) (pMAL-c2x-napA) . The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture . The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself . CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori. Biochem J . 2005 Jan 7; {Epub ahead of print} Positive cooperative activity and dimerization of the isolated ABC-ATPase domain of HlyB from E.coli; Benabdelhak H et al.; The ATPase activity of the ABC-ATPase domain of the HlyB transporter is required for secretion of E . coli haemolysin via the type I pathway . Whilst ABC-transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear . In this study we have analyzed the HlyB-ABC-domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers . The ATPase activity of the isolated ABC-domain clearly demonstrated positive co-operativity with a Hill coefficient of 1.7 . Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately reduced binding of 8-azido-ATP . Inhibition of activity by increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence . Finally, ATPase activity was sensitive towards ortho-vanadate with an IC 50 value of 16 muM consistent with the presence of transient dimers during ATP hydrolysis . Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC-ATPase was only detected as a monomer as measured by ultracentrifugation or gel filtration . In contrast, in the absence of salt the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers . Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration even in the presence of salt . These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis. Epidemiol Infect, 2004 Dec, 132(6), 1039 - 48 Estimating transmission parameters of F4+ E . coli for F4-receptor-positive and -negative piglets: one-to-one transmission experiment; Geenen PL et al.; F4+ Escherichia coli is an important agent of post-weaning diarrhoea in piglets . Piglets that express an adhesion site for F4+ E . coli in their small intestine (F4R+) shed higher numbers of F4+ E . coli than piglets lacking this site (F4R-) . We hypothesized that F4R+ piglets are more infectious and more susceptible for F4+ E . coli . This implies that in populations with F4R+ and F4R- piglets, the transmission would be dependent on the frequency of both types of animals . To quantify the difference in infectiousness and susceptibility, a one-to-one transmission experiment was performed with 20 pairs consisting of one inoculated and one contact piglet . Based on the contact infections observed, transmission parameters were estimated with generalized linear models . F4R+ piglets were infectious for other piglets and the reproduction ratio (R0) for homogeneous F4R+ populations, that is the average number of secondary infections that one F4R+ pig will cause during its entire infectious period in a population of susceptible F4R+ individuals only, was estimated as 7.1 . F4R+ piglets were more susceptible than F4R- piglets and reducing the fraction of F4R+ piglets of a population will reduce transmission . It was calculated that in order to prevent major outbreaks of F4+ E . coli (R0 < 1), the fraction of F4R+ piglets must be lower than 0.14. J Biol Chem . 2005 Jan 5; {Epub ahead of print} A new tyrosyl radical on F208 as ligand to the diiron center in E . coli ribonucleotide reductase, mutant R2-Y122H: Combined X-ray diffraction, and EPR/ENDOR studies; Kolberg M et al.; The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins . In wild-type R2 of E . coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Y122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides . In this work, we characterize the mutant E . coli protein R2-Y122H, where the radical site is substituted with a histidine residue . The X-ray structure verifies the mutation . R2-Y122H contains a novel stable paramagnetic center which we name "H", and which we have previously proposed to be a diferric iron center with a strongly coupled radical, FeIIIFeIIIRo . Here we report a detailed characterization of center "H", using 1H/2H- 14N/15N- and 57Fe-ENDOR in comparison with the FeIIIFeIV intermediate "X" observed in the iron reconstitution reaction of R2 . Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine . As F208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue F208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1 . This work demonstrates, that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical. Zhonghua Xue Ye Xue Za Zhi, 2004 Nov, 25(11), 666 - 70 {Expression of human HSF in E . coli and its effects on mobilization of hematopoietic stem cells in rhesus monkeys.}; Qi CM et al.; OBJECTIVE: To study the expression of hHSF in E . coli and its effect on the mobilization of hematopoietic stem/progenitor cells . METHODS: The hHSF gene was obtained by overlapping PCR and cloned into the vector pET30a to yield pET30a-hHSF, which was transformed into E . coli BL21(DE3) and expressed with IPTG induction . Subsequently, rhHSF was purified by gel filtration and cation exchange chromatography and subjected to refolding . Molecular weight of hHSF was measured by MALDI-TOF Mass Spectroscopy . The N terminal amino acid sequence rhHSF was determined by protein sequencing . rhHSF was profiled in rhesus monkey for mobilization of peripheral blood stem cells . Eight rhesus monkeys were equally divided into two groups . The first group was administered single subcutaneous injection of 500 microg/kg hHSF, while the other one was administered 10 microg.kg(-1).d(-1) G-CSF for 4 days followed by a single subcutaneous injection of 500 microg/kg rhHSF . RESULTS: The sequence coding hHSF was confirmed by sequencing and the induced-expression level was about 30% of total cell proteins . The purity of target protein was over 95% . The sequence of N terminal 10 amino acids and the amino acid composition were consistent with the theoretical parameters; molecular weight of rhHSF was 7540 . The peripheral CD34(+) cells, CFU-GM yields, and neutrophils peaked at 3 h (16.3-folds increase compared with baseline), 1 h (1.9-folds increase) and 45 min (4.4-folds increase) respectively after the single injection of rhHSF . The addition of rhHSF after the last dose of G-CSF boosted these levels to 25.8-folds, 8.7-folds and 8.3-folds respectively . CONCLUSION: hHSF is highly expressed in E . coli and rapidly mobilizes the hematopoietic stem/progenitor cells and neutrophils in rhesus monkeys . hHSF shows distinct synergistic effect with G-CSF. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005 Jan, 21(1), 33 - 6 {The fusion construction of HIV-1 Tat gene and efficient expression in E.coli.}; Lin JP et al.; AIM: To express high-level the Tat protein in E.coli . METHODS: Full-length HIV-1 Tat gene was amplified artificially by PCR and Tat gene was mutated site-specifically (substitution the codons AAG encoding the lysine at the 28th and the 50th site by the CAG encoding glutamine) in order to eliminate the transcriptional activity of Tat protein . The site-mutated Tat gene was fused with chaperone10 gene, and then was subcloned into vector pET28a . The recombinant plasmid was expressed in E.coli BL21(DE3) . The expressed products were identified by Western blot . RESULTS: Full-length HIV-1 Tat gene was amplified successfully by three rounds of PCR . The recombinant plasmid pET28a-chaperone 10-Tat was expressed efficiently in E.coli BL21(DE3) . Western blot analysis showed the expressed Tat fusion protein with relative molecular mass (M(r)) 24 000 could bind to anti-His-tag monoclonal antibody . CONCLUSION: Full-length HIV-1 Tat gene was cloned and chaperone 10-Tat fusion protein was expressed efficiently in E.coli BL21(DE3), which will lay the foundation for researching the pathogenic effect of HIV-1 Tat on AIDS. Biosens Bioelectron, 2005 Feb 15, 20(8), 1662 - 7 A high density microelectrode array biosensor for detection of E . coli O157:H7; Radke SM et al.; A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7 . The biosensor was fabricated from (100) silicon with a 2mum layer of thermal oxide as an insulating layer, an active area of 9.6mm(2) and consists of an interdigitated gold electrode array . The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface . Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples . The change in impedance caused by the bacteria was measured over a frequency range of 100Hz-10MHz . The biosensor was evaluated for E . coli O157:H7 detection in pure culture and inoculated food samples . The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples. Biochem Biophys Res Commun, 1976 Dec 20, 73(4), 881 - 4 Inhibition of E . coli beta-hydroxydecanoyl thioester dehydrase by ppGpp; Stein JP Jr et al.; Guanosine-5'-diphosphate-3'-diphosphate was found to inhibit beta-hydroxydecanoyl thioester dehydrase of E . coli at concentrations corresponding to those generated in vivo during amino acid starvation of rel+ cells. Zhonghua Xue Ye Xue Za Zhi, 1997 Sep, 18(9), 460 - 3 {Construction and expression in E . coli of single-chain F v fragment of antihuman activated platelets monoclonal antibody}; Wan H et al.; OBJECTIVE: In order to reduce the immunogenicity of murine monoclonal antibody (MAb) to human beings . METHODS: The cDNA encoding the variable regions of the mouse MAb SZ-51, which is specific against alpha-granule membrane protein(GMP) 140 on activated human platelets, was attached to the oligonucleotide of the linker peptide (Gly4 Ser)3 by means of recombinant DNA technique . The phagemid construct pHEN1-51scFv was obtained and introduced into the non-suppressor E . coli strain HB2151 for expression . RESULTS AND CONCLUSION: The expressed product could fold up into a soluble single-chain Fv fragment (scFv) and was released into the conditioned medium . The SZ-51scFv was proved to bind specifically to GMP140 by Western blot. Zhonghua Xue Ye Xue Za Zhi, 1997 Dec, 18(12), 634 - 7 {High expression of human thrombopoietin mutant fusion protein in E . coli}; Tian S et al.; OBJECTIVE: To obtain human thrombopoietin (Tpo)mutant cDNA encoding mature peptide N terminal 1 approximately 196 amino acids and investigate its expression in E . coli JM109 . METHODS: Polymerase chain reaction and DNA recombination techniques were employed . PCR product was inserted into pUC19 vector and sequenced and then cloned into expression vector pMAL-c2 . RESULTS: E . coli JM109 cells with plasmid pMAL -MBP/TpoM were induced by IPTG for 4-5 hours . SDS-PAGE analysis showed that MBP/TpoM molecular weight is about 63KD . Scanning analysis indicated that expressed protein accounts up to 37% of total E . coli proteins . CONCLUSION: The Tpo mutant of interest is successfully expressed in E . coli JM109 cells. Drug Metab Pharmacokinet, 2003, 18(1), 42 - 7 Large-scale Production of Genetically Engineered CYP3A4 in E . coli: Application of a Jarfermenter; Kanamori Y et al.; To produce a large amount of CYP3A4, we applied a jarfermenter (ABLE, BMJ-PI or BMS-PI) to culture the genetically engineered E . coli cells harboring CYP3A4 along with NADPH-cytochrome P450 reductase (OR) . The jarfermenter is a stirred bacterial culture vessel in which the pH, the dissolved oxygen (DO) and the temperature of a culture medium can be controlled . The expression of CYP3A4 in E . coli cells in the 500 mL of culture medium contained in the BMJ-PI (1 L vessel) (JFM-1) was examined by altering the parameters mentioned above . The highest expression of CYP3A4 in E . coli cells was attained when cultured at pH 6.0, at 30 degrees C under the DO of 0.1 ppm . The incubation was performed 18 hr after the addition of 1.5 mM isopropyl beta-D(-)-thiogalactopyranoside . The expression levels of CYP3A4 and the OR in the membrane fraction of E . coli cells were 267 nmol/L culture and 552 units/L culture, respectively . The CYP3A4 level was about three times higher than that obtained by incubation in a 500 mL flask (100 mL of medium) (84 nmol/L culture) . The testosterone 6beta-hydroxylase activity of CYP3A4 expressed in the membrane fraction of E . coli obtained with the JFM-1 was examined . The apparent K(m) and V(max) values were 66.4muM and 57.8 nmol/min/nmol CYP, respectively.Expecting the mass production of the CYP3A4 by a culture of E . coli, the possibility of a scale up of the culture with the BMS-PI (10 L vessel) (JFM-10) was examined . The optimal culture condition to achieve the highest expression of CYP3A4 with JFM-1 was employed . The expression levels of CYP3A4 and the OR obtained with JFM-1 and JFM-10 were almost equal . The total level of CYP3A4 obtained by using JFM-10 (5 L of medium) was calculated to be about 1.4 mumol.Based on these results, we confirm that the jarfermenter is a useful tool to produce large amounts of CYP3A4. Chem Commun (Camb), 2005 Jan 7, (1), 89 - 91 Epub 2004 Nov 25. NMR and ion selective electrode studies of hydraphile channels correlate with biological activity in E . coli and B . subtilis; Leevy WM et al.; Previous sodium ion transport results obtained by NMR methods are compared with those obtained by a new ISE technique and are found to correlate well with each other and with the biological activity against E . coli and B . subtilis. J Appl Microbiol, 2005, 98(1), 245 - 52 Assessment of resistance to colicinogenic Escherichia coli by E . coli O157:H7 strains; Schamberger GP et al.; Abstract g.p . schamberger and f . diez-gonzalez . 2004.Aims: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E . coli developed as a probiotic for use in cattle . Methods and Results: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development . Thirteen of 14 previously characterized colicinogenic E . coli strains were able to inhibit 74 serotype O157:H7 strains . Thirteen E . coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes . PCR products for colicins B, E-type, Ia/Ib and M were also detected . During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E . coli strains was observed, but rarely against multiple-colicinogenic strains . The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment . Conclusions: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely . Colicin D is the predominant colicin produced by colicinogenic E . coli O157:H7 strains . Significance and Impact of the Study: The potential for resistance development against colicin-based strategies for E . coli O157:H7 control may be very limited if more than one colicin type is used. Biotechnol Lett, 2004 Oct, 26(19), 1501 - 4 High level production of bovine angiogenin in E . coli by an efficient refolding procedure; Jang SH et al.; Recombinant bovine angiogenin (rbAng) was expressed in E . coli at up to 30% of total cell proteins but was produced as inclusion bodies . By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding . After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture . The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk . Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes. APMIS, 2004 Sep, 112(9), 569 - 84 Designation of O174 and O175 to temporary O groups OX3 and OX7, and six new E . coli O groups that include Verocytotoxin-producing E . coli (VTEC): O176, O177, O178, O179, O180 and O181; Scheutz F et al.; Two temporary Escherichia coli O group strains OX3 and OX7 are given permanent status as O174 and O175, respectively . Both these test strains were originally isolated from cases of human diarrhoea . Whereas the O174 strain is negative for known virulence genes, the O175 strain is positive with the probe derived from the CVD432 plasmid associated with the aggregative adherence phenotype, the Enteroaggregative heat-stable enterotoxin 1 gene (astA) and daaC (F1845 afimbrial adhesin) associated with the diffuse adherence (DA) phenotype . Additionally, six E . coli strains are established as antigenic test strains for six new O groups, designated O176, O177, O178, O179, O180 and O181 . All six strains produced Verocytotoxin and were positive for vtx1, vtx2, or both genes . Additional virulence genes associated with diarrhoeal disease in humans were found in four of the strains . O176 and O177 strains were isolated from calves, O178 and O181 strains from meat, the O179 strain was from human bloody diarrhoea, and the O180 strain from swine . Preliminary data on the occurrence and epidemiology of these eight new O groups amongst groups of diarrhoeagenic E . coli are reviewed. J Mol Evol, 2004 Dec, 59(6), 806 - 14 Higher gene duplicabilities for metabolic proteins than for nonmetabolic proteins in yeast and E . coli; Marland E et al.; Although the evolutionary significance of gene duplication has long been appreciated, it remains unclear what factors determine gene duplicability . In this study we investigated whether metabolism is an important determinant of gene duplicability because cellular metabolism is crucial for the survival and reproduction of an organism . Using genomic data and metabolic pathway data from the yeast (Saccharomyces cerevisiae) and Escherichia coli, we found that metabolic proteins indeed tend to have higher gene duplicability than nonmetabolic proteins . Moreover, a detailed analysis of metabolic pathways in these two organisms revealed that genes in the central metabolic pathways and the catabolic pathways have, on average, higher gene duplicability than do other genes and that most genes in anabolic pathways are single-copy genes. Environ Health Perspect, 2004 Nov, 112(16), 1614 - 21 The TAO-Gen algorithm for identifying gene interaction networks with application to SOS repair in E . coli; Yamanaka T et al.; One major unresolved issue in the analysis of gene expression data is the identification and quantification of gene regulatory networks . Several methods have been proposed for identifying gene regulatory networks, but these methods predominantly focus on the use of multiple pairwise comparisons to identify the network structure . In this article, we describe a method for analyzing gene expression data to determine a regulatory structure consistent with an observed set of expression profiles . Unlike other methods this method goes beyond pairwise evaluations by using likelihood-based statistical methods to obtain the network that is most consistent with the complete data set . The proposed algorithm performs accurately for moderate-sized networks with most errors being minor additions of linkages . However, the analysis also indicates that sample sizes may need to be increased to uniquely identify even moderate-sized networks . The method is used to evaluate interactions between genes in the SOS signaling pathway in Escherichia coli using gene expression data where each gene in the network is over-expressed using plasmids inserts. Nucleosides Nucleotides Nucleic Acids, 2004, 23(11), 1751 - 65 Side-chain conformational restriction in template-competitive inhibitors of E . coli DNA polymerase I Klenow fragment: synthesis, structural characterization and inhibition activity; Doughty MB et al.; Nucleotide triphosphate alpha-(4-azidophenyl)-1,N6-etheno-dATP 3 and its monophosphate 3m were synthesized by condensation of 2-halo-2-(4-azidophenyl)acetaldehyes with dATP and dAMP, respectively . Structure analysis shows that the azidophenyl side chain is attached to the alpha-position of the etheno ring (i.e., the carbon attached to N1 of the purine), and conformation calculations show minima in the etheno-phenyl bond rotation at 50 and 130 degrees where the bulk of the phenyl ring projects out from the plane of the etheno group . Like DNA Pol inhibitor 2-(4-azidophenacyl)thio-2'-deoxyadenosine 5'-triphosphate 1, nucleotide 3 is a template-competitive DNA polymerase inhibitor (TCPI), with a competitive Ki for Pol I KF of 3.41 microM, but has only weak activity as an HIV RT inhibitor relative to the template-competitive reverse transcriptase inhibitor 2-(4-azidophenacyl)thio-1,N6-etheno-2'-deoxyadenosine 5'-triphosphate 2 . Additionally, 3 photoinactivates KF in a time-dependent manner, confirming the kinetic data that 3 binds to the free form of KF . The TCPI activity of 3 provides evidence for an extended side chain conformational preference in the combined substrate polymerase inhibitors. J Mol Model (Online), 2004 Dec, 10(5-6), 317 - 27 Epub 2004 Aug 27. Modeling the E . coli 4-hydroxybenzoic acid oligoprenyltransferase ( ubiA transferase) and characterization of potential active sites; Brauer L et al.; 4-Hydroxybenzoate oligoprenyltransferase of E . coli, encoded in the gene ubiA, is an important key enzyme in the biosynthetic pathway to ubiquinone . It catalyzes the prenylation of 4-hydroxybenzoic acid in position 3 using an oligoprenyl diphosphate as a second substrate . Up to now, no X-ray structure of this oligoprenyltransferase or any structurally related enzyme is known . Knowledge of the tertiary structure and possible active sites is, however, essential for understanding the catalysis mechanism and the substrate specificity.With homology modeling techniques, secondary structure prediction tools, molecular dynamics simulations, and energy optimizations, a model with two putative active sites could be created and refined . One active site selected to be the most likely one for the docking of oligoprenyl diphosphate and 4-hydroxybenzoic acid is located near the N-terminus of the enzyme . It is widely accepted that residues forming an active site are usually evolutionary conserved within a family of enzymes . Multiple alignments of a multitude of related proteins clearly showed 100% conservation of the amino acid residues that form the first putative active site and therefore strongly support this hypothesis . However, an additional highly conserved region in the amino acid sequence of the ubiA enzyme could be detected, which also can be considered a putative (or rudimentary) active site . This site is characterized by a high sequence similarity to the aforementioned site and may give some hints regarding the evolutionary origin of the ubiA enzyme.Semiempirical quantum mechanical PM3 calculations have been performed to investigate the thermodynamics and kinetics of the catalysis mechanism . These results suggest a near S(N)1 mechanism for the cleavage of the diphosphate ion from the isoprenyl unit . The 4-hydroxybenzoic acid interestingly appears not to be activated as benzoate anion but rather as phenolate anion to allow attack of the isoprenyl cation to the phenolate, which appeared to be the rate limiting step of the whole process according to our quantum chemical calculations . Our models are a basis for developing inhibitors of this enzyme, which is crucial for bacterial aerobic metabolism.FIGURE Structure of the model of ubiA oligoprenyltransferase derived from the photosynthetic reaction center (1PRC) . Putative active amino acid residues and substrates are shown as capped sticks to describe their location and geometry in the putative active sites . The violet spheres identify Mg(2+). Inflamm Res, 2004 Oct, 53(10), 551 - 5 Low levels of antibodies against E . coli and mycobacterial 65kDa heat shock proteins in patients with inflammatory bowel disease; Huszti Z et al.; OBJECTIVE AND DESIGN: The aim of the present study was to support and extend our initial observation, where we found low levels of antibodies against mycobacterial 65kD heat shock proteins in patients with inflammatory bowel disease (IBD) . For this purpose we tested a new group of 124 patients with IBD, and beside measuring antibodies to Mycobacterium bovis 65kD heat shock protein (Hsp65) and human 60kD heat shock protein (Hsp60) as described previously, we also determined IgG antibody levels to Hsp65 from E . coli, called GroEL . PATIENTS AND CONTROL SUBJECTS: seventy-four patients with Crohn's disease (CD) (30 males, 44 females, 33 (27-45) years old, median (interquartile range)) and 50 patients with ulcerative colitis (UC) (22 males, 28 females, 38 (30-50) years old) were involved in the study . 110 healthy subjects (34 males, 76 females, 47 (37-53) years old) served as controls . Study subjects were consecutive patients referred to an IBD center for complex treatment of the disease . Methods and statistical analysis: The amounts of IgG-type antibodies reacting with proteins of the chaperonin 60 family were assessed by ELISA . Since the antibody levels to heat-shock proteins as variables were not normally distributed, non-parametric Mann-Whitney test and Dunn post hoc test were used for group comparisons . RESULTS: Median levels of anti-GroEL (7,5 (3,5-18,3)) and anti-Hsp65 (4,8 (2,1-7,85)) were significantly (GroEL p = 0,008; and Hsp65 p < 0,001) lower in the IBD patients than in the healthy subjects (GroEL: 10,0 (5,4-31,0); Hsp65: 7,04 (4,66-12,77)) . However this difference was found to be restricted to the CD patients (GroEL: 7,5 (3,7-14,2); p < 0,05; Hsp65: 4,35 (1,90-6,94); p < 0,001) . We did not find difference in the concentration of anti-human Hsp60 IgG levels between patients (Hsp60: 45,5 (24,9-69,0)) and healthy controls (38,4 (21,6-69,4) . Regarding the serum concentrations of each antibody tested there was no significant difference between the active and inactive stage of disease . CONCLUSION: Our present findings support conclusion of our previous work, antibody levels not only for Mycobacterium bovis hsp65 but for E . coli GroEl were found to be decreased as well . In contrast no changes in the concentrations of human anti-hsp60 antibodies were observed . These findings indicate that production of antibodies to 65 kDa bacterial heat shock proteins is selectively impaired in IBD. BMC Microbiol . 2004 Dec 08;4(1):47. Allele specific synthetic lethality between priC and dnaAts alleles at the permissive temperature of 30 degrees C in E . coli K-12; Hinds T et al.; BACKGROUND: DnaA is an essential protein in the regulation and initiation of DNA replication in many bacteria . It forms a protein-DNA complex at oriC to which DnaC loads DnaB . DNA replication forks initiated at oriC by DnaA can collapse on route to the terminus for a variety of reasons . PriA, PriB, PriC, DnaT, Rep and DnaC form multiple pathways to restart repaired replication forks . DnaC809 and dnaC809,820 are suppressors of priA2::kan mutant phenotypes . The former requires PriC and Rep while the latter is independent of them . RnhA339::cat mutations allow DnaA-independent initiation of DNA replication . RESULTS: It is shown herein that a priC303::kan mutation is synthetically lethal with either a dnaA46 or dnaA508 temperature sensitive mutation at the permissive temperature of 30 degrees C . The priC-dnaA lethality is specific for the dnaA allele . The priC303::kan mutant was viable when placed in combination with either dnaA5, dnaA167, dnaA204 or dnaA602 . The priC-dnaA508 and priC-dnaA46 lethality could be suppressed by rnhA339::cat . The priC-dnaA508 lethality could be suppressed by a dnaC809,820 mutation, but not dnaC809 . Neither of the dnaC mutations could suppress the priC-dnaA46 lethality . CONCLUSIONS: A hitherto unknown function for either DnaA in replication restart or PriC in initiation of DNA replication that occurs in certain dnaA temperature sensitive mutant strains at the permissive temperature of 30 degrees C has been documented . Models considering roles for PriC during initiation of DNA replication and roles for DnaA in replication restart were tested and found not to decisively explain the data . Other roles of dnaA in transcription and nucleoid structure are additionally considered. FEBS Lett, 2004 Dec 3, 578(1-2), 163 - 8 Functional properties of the alternative NADH:ubiquinone oxidoreductase from E . coli through comparative 3-D modelling; Schmid R et al.; The alternative NADH:ubiquinone oxidoreductase (NDH-2) from Escherichia coli is a membrane protein playing a prominent role in respiration by linking the reduction of NADH to the quinone pool . Remote sequence similarity reveals an evolutionary relation between alternative NADH:quinone oxidoreductases and the SCOP-family "FAD/NAD-linked reductases" . We have created a structural model for NDH-2 from E . coli through comparative modelling onto a template from this family . Combined analysis of our model and sequence conservation allowed us to include the cofactor FAD and the substrate NADH in atomic detail . Furthermore, we propose the most plausible orientation of NDH-2 relative to the membrane and specify a region of the protein potentially involved in ubiquinone binding. J Gene Med . 2004 Dec 6; {Epub ahead of print} Transcriptional tumor-selective promoter targeting of E . coli purine nucleoside phosphorylase for pancreatic cancer suicide gene therapy; Deharvengt S et al.; BACKGROUND: Pancreatic cancer remains a rapidly fatal disease . Suicide gene therapy has been shown to be an effective tool for pancreatic tumor cell destruction, but a cell-specific gene delivery is required to limit host toxicity . The objective of this study was both to design recombinant vectors in which the suicide gene E . coli purine nucleoside phosphorylase (ePNP) is under the control of either CEA or MUC1 promoter sequences and to investigate on experimental pancreatic carcinomas the selective killing effects of the conditional ePNP/prodrug (MePdR) system . METHODS: Transcriptional activities of CEA and MUC1 promoter sequences were analyzed using luciferase reporter gene constructions . Thereafter, recombinant vectors expressing ePNP under control of the most promising pCEA and pMUC1 sequences were designed and used to establish stable tumor cell transfectants from two human pancreatic cell lines, respectively tumor-marker positive (BxPc3) or negative (Panc-1), then applied for in vitro and in vivo experiments . RESULTS: Transient experiments indicated that CEA and MUC1 promoter sequences confer specificity while preserving high transcriptional activities . The MePdR treatment induced a high in vitro cytotoxicity on the sole CEA- and MUC1-producing cell lines (i.e . BxPc3-CEA and -MUC1/ePNP) . In the same way, prodrug treatment induced a significant tumor regression on the sole tumor-marker-positive BxPc3 xenografts, whilst the Panc1-CEA and -MUC1/ePNP tumors were not affected . CONCLUSIONS: These data confirm and extend the antitumor efficacy of the ePNP/MePdR killing system and demonstrate the feasibility of the transcriptional targeting strategy under tumor marker promoter control and thereby a preferential killing of CEA- and MUC1-producing pancreatic tumor cells . Thus, efficient in vivo gene delivery and transcriptional targeting constitute the major future clinical challenge for a selective pancreatic cancer suicide gene strategy . Copyright (c) 2004 John Wiley & Sons, Ltd. J Mol Biol, 2004 Dec 10, 344(5), 1287 - 309 Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA; Fischer CJ et al.; Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA . Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA . Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt . We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality . Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation . We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C) . However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro . DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA. Mem Inst Oswaldo Cruz, 2004 Oct, 99(6), 545 - 52 Epub 2004 Oct. Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E . coli O serogroups--a review; Campos LC et al.; The so called enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E . coli, enterotoxigenic E . coli, and enteroaggregative E . coli . The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheagenic categories . Their adherence patterns and genetic relationships are also presented . The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in Sao Paulo from children with diarrhea between 1970 and 1990 . Since some O serogroups include more than one diarrheagenic category O serogrouping only should be abandoned as a diagnostic method . However serotyping is a reliable method for those serotypes that correspond to clones. Infect Immun, 2004 Dec, 72(12), 7282 - 93 Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E . coli O157:H7; Zhang L et al.; Enterohemorrhagic Escherichia coli (EHEC) O157:H7 subverts host cells through a type III secretion system encoded by the locus for enterocyte effacement (LEE) . Genome sequencing of this pathotype revealed the existence of a gene cluster encoding components of a second cryptic type III secretion system, E . coli type III secretion system 2 (ETT2) . Recently, we showed that the ETT2 gene cluster is present in whole or in part in the majority of E . coli strains but is unable to encode a functional secretion system in most strains, including EHEC O157:H7 . However, here we show that mutational inhibition of two regulatory genes (ECs3720 or etrA and ECs3734 or eivF) from the ETT2 cluster in EHEC O157:H7 leads to greatly increased secretion of proteins encoded by the LEE and to increased adhesion to human intestinal cells . Studies in which transcriptional fusions and microarrays were used indicated that EtrA and EivF exert profound negative effects on gene transcription within the LEE . Consistent with these observations, expression of these regulators in an EHEC O26:H- strain led to suppression of protein secretion under LEE-inducing conditions . These findings provide fresh examples of the influence of mobile genetic elements on regulation of the LEE and of cross talk between type III secretion system gene clusters . In addition, they provide a cautionary tale because they show that the effects of regulatory genes can outlive widespread decay of other genes in a functionally coherent gene cluster, a phenomenon that we have named the "Cheshire cat effect." It also seems likely that variations in the ETT2 regulator repertoire might account for strain-to-strain variation in secretion of LEE-encoded proteins. J Biol Chem . 2004 Nov 22; {Epub ahead of print} Promiscuity in the geometry of electrostatic interactions between the E . coli Mdr transporter MdfA and cationic substrates; Adler J et al.; The Escherichia coli multidrug transporter MdfA contains a single membrane-embedded charged residue (E26) that plays a critical role in recognition of cationic substrates (Edgar, R . and Bibi, E . 1999 EMBO J . 18, 822-832) . Using an inactive mutant (MdfA-E26T), we isolated a spontaneous second-site mutation (MdfA-E26T/V335E) that re-established recognition of cationic drugs by the transporter . Only a negative charge at position 335 was able to restore the functioning of the inactive mutant MdfA-E26T . Intriguingly, the two genetically interacting residues are located at remote and distinct regions along the secondary structure of MdfA: E26 is located in the periplasmic half of transmembrane helix 1, and as shown here, the complementing charge at position 335 resides within the cytoplasmic loop connecting transmembrane helices 10 and 11 . The spatial relation between the two residues was investigated by cross-linking . A functional split version of MdfA devoid of cysteines was constructed and introduced with a cysteine-pair at positions 26 and 335 . Strikingly, the results indicate that residues 26 and 335 are spatially adjacent, suggesting that they both constitute parts of the multidrug recognition pocket of MdfA . The fact that with cationic substrates electrostatic interactions are preserved even if the critical acidic residue is placed on another face of the pocket reveals an additional dimension of promiscuity in multidrug recognition and transport. Protein Expr Purif, 2004 Dec, 38(2), 184 - 95 High-level production of recombinant sulfide-reactive hemoglobin I from Lucina pectinata in Escherichia coli . High yields of fully functional holoprotein synthesis in the BLi5 E . coli strain; Leon RG et al.; Hemoglobin I (HbI) from Lucina pectinata is a monomeric protein composed of 143 amino acids with high sulfide affinity . Its unique heme pocket contains three residues not commonly found in vertebrate globins: Phe 29 (B10), Gln 64 (E7), and Phe 68 (E11), which are thought to be important for high affinity for hydrogen sulfide . Recombinant HbI (rHbI) and several site-directed mutants were cloned and expressed in Escherichia coli yielding high amounts of protein . The highest rHbI protein yield was obtained when the HbI cDNA was cloned into the pET28 (a+) expression vector, transformed into BLi5 cells, the induction performed with 1 mM IPTG at 30 degrees C and TB medium was supplemented with 30 microg/mL hemin chloride and 1% glucose . The highest yield obtained of HbI was 32 mg/L of culture using Fernbach flasks . UV/Visible spectral analysis showed that rHbI binds heme and ESI-MS shows that its molecular weight corresponds to the expected size . Kinetic studies with H2S confirmed that rHbI and HbI have identical binding properties, where the kON for the clam's Hb is 2.73x10(4)M-1s-1 and for rHbI is 2.43x10(4)M-1s-1. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Nov, 20(6), 682 - 5 {Study on mimotopes of E.coli lipopolysaccharide 2630.}; Luo HB et al.; AIM: To screen mimotopes of E.coli lipopolysaccharide(LPS) 2630 from c7c phage display peptide library . METHODS: The LPS mimotopes were screened from c7c phage display peptide library by using affinity chromatograph-purified polyclonal antibody against E.coli LPS 2630(L2630), and the antigenicity of selected clones was identified by ELISA . RESULTS: After 3 rounds of biopanning, 12 out of 20 phage clones were identified as positive clones which could bind to polyclonal anti-L2630 antibody, and 5 of these 12 clones could bind to polyclonal anti-S.typhi LPS 7261(L7261) antibody . The deduced amino acid sequence analysis showed that 8 of 12 clones had the conservative sequence: X-DGLL-XX or X-EDGLL-X . CONCLUSION: The peptides displayed on these phage clones can mimic the epitopes of L2630, and 5 of these phage clones mimic the common epitopes of L2630 and L7261. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Nov, 20(6), 671 - 4 {Construction of recombinant plasmid pYTA/Abeta-HBcAg and its expression in E.coli.}; Feng GF et al.; AIM: To study the expression of fusion gene Abeta-HBcAg in E.coli and detect the immunogenicity of fusion protein Abeta-HBcAg . METHODS: The Abeta-HBcAg fusion gene was cut from recombinant plasmid pBV220/Abeta-HBcAg, and was inserted into the plasmid pYTA1 to construct recombinant plasmid pYTA/Abeta-HBcAg . The recombinant plasmid was transformed into E.coli DH5alpha, and expressed under IPTG induction . The expression of the fusion protein Abeta-HBcAg was detected by SDS-PAGE . The antigenicity of the fusion protein was detected by ELISA . BALB/c mice were immunized intraperitoneally with the fusion protein purified by salting out with saturate ammonium sulfate . The titers of anti- Abeta antibodies of the immunized mice was evaluated by ELISA . RESULTS: Fusion protein existed in supernatant of the bacteria lysate and its expression level was about 7% of the total bacteria protein . The fusion protein reacted with both Abeta and HBcAg . The highest titer of anti-Abeta antibodies could reach to 1:16 000 after immunization for 3 times . CONCLUSION: Recombinant gene Abeta-HBcAg can be expressed in E.coli DH5alpha . The expression protein exists in supernatant of the bacteria lysate and has good immunogenicity . This study lays the foundation for the experimental animal study of AD gene-engineering vaccine. J Biol Chem . 2004 Nov 18; {Epub ahead of print} Peptidase activity of the E . coli HSP31 chaperone; Malki A et al.; Hsp31, the Escherichia coli hcha gene product is a molecular chaperone whose activity is inhibited by ATP at high temperature . Its crystal structure reveals a putative Cys184, His185 and Asp213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity . A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity towards bovine serum albumin and a peptidase activity towards two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear . In this study, we report that Hsp31 does not diplay any significant proteolytic activity, but has peptidolytic activity . The aminopeptidase cleavage preference of Hsp31 is Ala>Lys>Arg>His, suggesting that Hsp31 is an aminopeptidase of broad specificity . Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide, and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase . The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, concording with the importance of the putative His85, His122 and Glu90 metal binding site revealed by crystallographic studies . An Hsp31-deficient mutant accumulates more 8-12mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro . Proteins interacting with Hsp31 have been identified by reverse purification of a crude E . coli extract on a Hsp31-affinity column, followed by SDS polyacrylamide electrophoresis and mass spectrometry . The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu and tryptophanase were all found to interact with Hsp31, thus substantiating this latter's role as both chaperone and peptidase. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 55 - 66 Membrane integration of E . coli model membrane proteins; Facey SJ et al.; The molecular events of membrane translocation and insertion have been investigated using a number of different model proteins . Each of these proteins has specific features that allow interaction with the membrane components which ensure that the proteins reach their specific local destination and final conformation . This review will give an overview on the best-characterized proteins studied in the bacterial system and emphasize the distinct aspects of the pathways. Genes Dev, 2004 Nov 15, 18(22), 2812 - 21 A chaperone network controls the heat shock response in E . coli; Guisbert E et al.; The heat shock response controls levels of chaperones and proteases to ensure a proper cellular environment for protein folding . In Escherichia coli, this response is mediated by the bacterial-specific transcription factor, sigma32 . The DnaK chaperone machine regulates both the amount and activity of sigma32, thereby coupling sigma32 function to the cellular protein folding state . In this manuscript, we analyze the ability of other major chaperones in E . coli to regulate sigma32, and we demonstrate that the GroEL/S chaperonin is an additional regulator of sigma32 . We show that increasing the level of GroEL/S leads to a decrease in sigma32 activity in vivo and this effect can be eliminated by co-overexpression of a GroEL/S-specific substrate . We also show that depletion of GroEL/S in vivo leads to up-regulation of sigma32 by increasing the level of sigma32 . In addition, we show that changing the levels of GroEL/S during stress conditions leads to measurable changes in the heat shock response . Using purified proteins, we show that that GroEL binds to sigma32 and decreases sigma32-dependent transcription in vitro, suggesting that this regulation is direct . We discuss why using a chaperone network to regulate sigma32 results in a more sensitive and accurate detection of the protein folding environment. Colloids Surf B Biointerfaces, 2004 Nov 25, 39(1-2), 45 - 51 E . coli adhesion to silica in the presence of humic acid; Parent ME et al.; The influence of humic acid on the adhesion of Escherichia coli to silica particles or glass surfaces was investigated . After adsorbing various amounts of humic acid to the particles or surfaces, bacteria were added to the sample and allowed to adhere . For the silica particles the number of bacteria-particle couplets formed were counted from video microscopy images . For the glass surfaces, a differential electrophoresis force was applied, and the force required to detach the bacteria was quantified . These experiments showed a slight increase in the number of couplets formed in the presence of humic acid, and also showed a slight increase in the force required for detachment of the bacteria . Although an increase in adhesion number and strength was measured, the magnitude of the increase was small, indicating that humic acid plays a small role in bacterial adhesion to silica or glass surfaces. Trends Microbiol, 2004 Dec, 12(12), 569 - 76 Receptor clustering and signal processing in E . coli chemotaxis; Sourjik V; Chemotaxis in Escherichia coli is one of the most thoroughly studied model systems for signal transduction . Receptor-kinase complexes, organized in clusters at the cell poles, sense chemoeffector stimuli and transmit signals to flagellar motors by phosphorylation of a diffusible response regulator protein . Despite the apparent simplicity of the signal transduction pathway, the high sensitivity, wide dynamic range and integration of multiple stimuli of this pathway remain unexplained . Recent advances in computer modeling and in quantitative experimental analysis suggest that cooperative protein interactions in receptor clusters play a crucial role in the signal processing during bacterial chemotaxis. Biochim Biophys Acta, 2004 Nov 18, 1675 1-3, 62 - 70 Expression of an immunologically reactive merozoite surface protein (MSP-1(42)) in E . coli; Leung WH et al.; The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1(42)) is one of the anti-malarial vaccine candidate antigens . In the present study, recombinant MSP-1(42) was expressed as a fusion protein in a novel E . coli host . The average yield of the recombinant protein was 48 mg/l of bacterial culture . The antigenicity and immunogenicity of the purified protein were evaluated by comparing the results with those obtained from a well-characterized recombinant MSP-1(42) (Bmp42) expressed in the baculovirus expression system previously described from our laboratory . We observed that there is a high degree of similarities between the two recombinant proteins . Based on the results from T and B cell response, in vitro parasite growth inhibition, as well as cross-reactivities with several well-characterized MSP-1 specific Mabs, the bacterial expressed protein is apparently comparable to Bmp42 in terms of immunoreactivities . Our results suggest that the bacterial expression system could be employed to express immunologically active recombinant MSP-1(42) at elevated levels . This system may be an attractive alternative for producing a protective vaccine for human use at lower cost. Vaccine, 2004 Nov 25, 23(2), 222 - 31 Plant-synthesized E . coli CFA/I fimbrial protein protects Caco-2 cells from bacterial attachment; Lee JY et al.; A DNA fragment encoding the cholera toxin A2 subunit (CTA2) linked to the enterotoxigenic Escherichia coli (ETEC) colony forming fimbrial antigen CFA/I was inserted into a plant expression vector containing the cholera toxin B subunit (CTB) fused to the rotavirus enterotoxin 22 amino acid epitope NSP422 . Anti-CFA/I antibodies recognized a single band of approximately 72-kDa in transformed potato tuber tissue consistent with CFA/I-CTA2 and CTB-NSP4 fusion protein assembly into a cholera holotoxin-like structure . Enzyme-linked immunosorbent assay (GM1 ELISA) indicated that the CFA/I-CTA2 fusion protein bound specific GM1 ganglioside membrane receptors and made up approximately 0.002% of the total soluble tuber protein . Oral immunization of BALB/c mice with transformed tuber tissues generated anti-CFA/I serum and intestinal IgG and IgA secretory antibodies . Attachment of ETEC H10407 to enterocyte-like Caco-2 human colon carcinoma cells incubated with antiserum from immunized mice was reduced by 15% in comparison with Caco-2 cells incubated with serum from unimmunized mice . Immunogold staining of bacterial preparations revealed deposition of gold particles on E . coli H10407 fimbria incubated with immune serum but not on fimbria treated with sera from unimmunized mice demonstrating the specificity of antibodies in the immune serum for binding to CFA/I protein containing fimbria . The protection against toxic E . coli binding to Caco-2 cells generated by antisera from mice immunized with plant-synthesized CFA/I antigen demonstrates the feasibility of plant-based multi-component vaccine protection against enterotoxigenic E . coli, rotavirus and cholera, three enteric diseases that together exert the highest levels of child morbidity and mortality in economically emerging countries. FEBS Lett, 2004 Nov 5, 577(1-2), 101 - 4 The P3 domain of E . coli ribonuclease P RNA can be truncated and replaced; Tanaka T et al.; We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre-tRNA substrate . The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre-tRNA substrate, but did affect the efficiency of cleavage of the substrate . Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E . coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme. Eur J Biochem, 2004 Nov, 271(21), 4204 - 12 Alternative substrates for wild-type and L109A E . coli CTP synthases: kinetic evidence for a constricted ammonia tunnel; Lunn FA et al.; Cytidine 5'-triphosphate (CTP) synthase catalyses the ATP-dependent formation of CTP from uridine 5'-triphosphate using either NH(3) or l-glutamine as the nitrogen source . The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH(3) that is generated is transferred via an NH(3) tunnel {Endrizzi, J.A., Kim, H., Anderson, P.M . & Baldwin, E.P . (2004) Biochemistry43, 6447-6463} to the active site of the N-terminal synthase domain where the amination reaction occurs . Replacement of Leu109 by alanine in Escherichia coli CTP synthase causes an uncoupling of glutamine hydrolysis and glutamine-dependent CTP formation {Iyengar, A . & Bearne, S.L . (2003) Biochem . J.369, 497-507} . To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH(3) tunnel, we examined the ability of wild-type and L109A CTP synthases to utilize NH(3), NH(2)OH, and NH(2)NH(2) as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, gamma-glutamyl hydroxamate, and gamma-glutamyl hydrazide, respectively . We show that the uncoupling of the hydrolysis of gamma-glutamyl hydroxamate and nascent NH(2)OH production from N(4)-hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase . These results suggest that the NH(3) tunnel of L109A, in the presence of bound allosteric effector guanosine 5'-triphosphate, is not leaky but contains a constriction that discriminates between NH(3) and NH(2)OH on the basis of size. Shi Yan Sheng Wu Xue Bao, 2004 Aug, 37(4), 269 - 75 {Cloning of potato invertase inhibitor St-inh cDNA and its expression in E . coli and functional analysis}; Cheng SH et al.; A full length cDNA clone encoding invertase inhibitor was isolated by RT-PCR combined with 5' RACE from potato (S . tuberosum) tubers of cv . JH and designated as St-inh . The encoding region of St-inh is of 663bp encoding a protein of 221 amino acids . The DNA fragment including St-inh cDNA was cloned into the vector pET28a (+) and expressed successfully in E . coli . Co-incubation of the proteins produced by St-inh in E . coli and the invertase extracts from potato tubers of cv . E1, JH and tomato fruits showed that the invertase activities of potato tubers and tomato fruits decreased by 34.3%, 21% and 33.8% respectively . These results indicated that products of St-INH protein had a function of invertase inhibitors . The analysises of the nucleotide and amino acid sequences using BLAST and T-COFFEE demonstrated that St-inh cDNA was of over 95% homologous to Kunitz-type C and there was a typical domain of Kunitz-type protein {L, I, V, M}-X-D-X-{E, D, N, T, Y-{D, G}-{R, K, H, D, E, N, Q}-X-{L, I, V, M}-X(5)-Y-X-{L, I, V, M . Therefore, it was conjectured that St-inh could be a member of Kunitz-type gene family. FEBS Lett, 2004 Oct 22, 576(3), 336 - 8 A novel method of analyzing proline synonymous codons in E . coli; Wang ML et al.; Proline is a special imino acid in protein and the isomerization of the prolyl peptide bond has notable biological significance and influences the final structure of protein greatly, so the correlation between proline synonymous codon usage and local amino acid, the correlation between proline synonymous codon usage and the isomerization of the prolyl peptide bond were both investigated in the Escherichia coli genome by using a novel method based on information theory . The results show that in peptide chain, the residue at the first position C-terminal influences the usage of proline synonymous codon greatly and proline synonymous codons contain some factors influencing the isomerization of the prolyl peptide bond. J Med Virol, 2004 Dec, 74(4), 641 - 9 E . coli-expressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability; Tan M et al.; The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction . We report here the application of the E . coli expression system in the production of recombinant NV capsid proteins . In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E . coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E . coli-expressed VA387 proteins did not form VLPs . Using the E . coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207 . Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors . VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus . Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells. J Mol Biol, 2004 Oct 29, 343(4), 931 - 42 Charge translocation during cosubstrate binding in the Na+/proline transporter of E.coli; Zhou A et al.; Charge translocation associated with the activity of the Na(+)/proline cotransporter PutP of Escherichia coli was analyzed for the first time . Using a rapid solution exchange technique combined with a solid-supported membrane (SSM), it was demonstrated that Na(+)and/or proline individually or together induce a displacement of charge . This was assigned to an electrogenic Na(+)and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k>50s(-1) which preceeds the rate-limiting step . Based on the kinetic analysis of our electrical signals, the following characteristics are proposed for substrate binding in PutP . (1) Substrate binding is electrogenic not only for Na(+), but also for the uncharged cosubstrate proline . The charge displacement associated with the binding of both substrates is of comparable size and independent of the presence of the respective cosubstrate . (2) Both substrates can bind individually to the transporter . Under physiological conditions, an ordered binding mechanism prevails, while at sufficiently high concentrations, each substrate can bind in the absence of the other . (3) Both substrate binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate . (4) Proline binding proceeds in a two-step process: low affinity (approximately 1mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition. Mutat Res, 2004 Nov 14, 564(1), 31 - 8 Comparative study of the antimutagenic potential of Vitamin E in different E . coli strains; Nikolic B et al.; The antimutagenic potential of Vitamin E due to its antioxidative properties was studied . The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action . The assay is composed of three tests . In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain . Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT) . In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability . To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test . Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E . coli K12 assay-system as well as in the WP2 antimutagenicity test . This study confirms that mismatch repair is essential for repair of oxidative DNA damage . The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases . Moreover, it can reduce microsatellite instability . After further validation, the new E . coli K12 assay-system can be used to test the antimutagenic potential of antioxidants. FEBS Lett, 2004 Oct 8, 576(1-2), 97 - 100 F(1)F(0) ATP synthase subunit c is targeted by the SRP to YidC in the E . coli inner membrane; van Bloois E et al.; Escherichia coli inner membrane proteins (IMPs) use different pathways for targeting and membrane integration . We have examined the biogenesis of the F1F0 ATP synthase subunit c, a small double spanning IMP, using complementary in vivo and in vitro approaches . The data suggest that F0c is targeted by the SRP to the membrane, where it inserts at YidC in a Sec-independent mechanism . F0c appears to be the first natural substrate of this novel pathway . FEBS Lett, 2004 Oct 8, 576(1-2), 81 - 5 Overproduction of CcmABCDEFGH restores cytochrome c maturation in a DsbD deletion strain of E . coli: another route for reductant? Stevens JM, Gordon EH, Ferguson SJ. The multidomain transmembrane protein DsbD is essential for cytochrome c maturation (Ccm) in Escherichia coli and transports reductant to the otherwise oxidising environment of the bacterial periplasm . The Ccm proteins ABCDEFGH are also essential and we show that the overproduction of these proteins can unexpectedly complement for the absence of DsbD in a deletion strain by partially restoring the production of an exogenous c-type cytochrome under aerobic and anaerobic conditions . This suggests that one or more of the Ccm proteins can provide reductant to the periplasm . The Ccm proteins do not, however, restore the normal disulfide mis-isomerisation phenotype of the deletion strain, as shown by assay of the multidisulfide-bonded enzyme urokinase . J Biochem Mol Biol, 2004 May 31, 37(3), 351 - 5 Rapid preparation of total nucleic acids from E . coli for multi-purpose applications; Cheng L et al.; Separate protocols are commonly used to prepare plasmid DNA, chromosomal DNA, or total RNA from E . coli cells . Various methods for the rapid preparation of plasmid DNA have been developed previously, but the preparation of the chromosomal DNA and total RNA are usually laborious . We report here a simple, fast, reliable, and cost-effective method to extract total nucleic acids from E . coli by direct lysis of the cells with phenol . Five distinct and sharp bands, which correspond to chromosomal DNA, plasmid DNA, 23S rRNA, 16S rRNA, and a mixture of small RNA, were observed when analyzing the prepared total nucleic acids on a regular 1-2% agarose gel . The simple and high-quality preparation of the total nucleic acids in a single tube allowed us to rapidly screen the recombinant plasmid, as well as to simultaneously monitor the change of the plasmid copy number and rRNA levels during the growth of E . coli in the liquid medium. J Mol Biol, 2004 Oct 22, 343(3), 569 - 87 A hydrophobic patch on the flap-tip helix of E.coli RNA polymerase mediates sigma(70) region 4 function; Geszvain K et al.; The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) to position it for recognition of the -35 element of promoters . We report that this function depends on a hydrophobic patch on one face of the short stretch of alpha helix located at the tip of the flap domain, called the flap-tip helix . Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and sigma region 4, as determined by protease sensitivity assays, and impaired transcription from -35-dependent promoters . We suggest that contact of the flap-tip helix hydrophobic patch to the sigma region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4 . This contact allowed region 4.2 recognition of the -35 promoter element and appeared to stabilize region 4 interaction with the beta' Zn(2+) binding domain . Our studies failed to detect any role for sigma region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1359 - 65 Coexpression of Vitreoscilla hemoglobin reduces the toxic effect of expression of D-amino acid oxidase in E . coli; Chien LJ et al.; Expression of the gene (daao) encoding D-amino acid oxidase (DAAO) in Escherichia coli typically results in a marked decrease of cell viability, and it has generally been assumed that the consumption of intracellular D-alanine by DAAO is responsible for this effect . Vitreoscilla hemoglobin (VHb) gene (vgb) was coexpressed with Rhodosporidium toruloides D-amino acid oxidase in E . coli BL21(DE3) and BL21(DE3)pLysS, expression hosts differing in the stringency of suppressing basal transcription . Not only was the toxic effect of DAAO on cell growth relieved but also the pronounced cell lysis of BL21(DE3)pLysS caused by the expression of DAAO was prevented by coexpressing VHb with DAAO . As a result of the higher cell density achieved, DAAO activity about 1.5-fold higher than that of DAAO-expressing control strains could be obtained by DAAO/VHb-coexpressing strains . The relieving effect of VHb on DAAO toxicity resulted from its oxygen-binding ability . The low availability of free intracellular oxygen reduced DAAO activity and consequently its toxicity. Bioinformatics . 2004 Sep 28; {Epub ahead of print} Improving promoter prediction for the NNPP2.2 algorithm: a case study using E-Coli DNA sequences; Burden S et al.; MOTIVATION: Although a great deal of research has been undertaken in the area of promoter prediction, prediction techniques are still not fully developed . Many algorithms tend to exhibit poor specificity, generating many false positives, or poor sensitivity . The neural network prediction program NNPP2.2 is one example . RESULTS: To improve the NNPP2.2 prediction technique, the distance between the transcription-start-site (TSS) associated with the promoter and the translation-start-site (TLS) of the subsequent gene coding region has been studied for E.Coli-K12 bacteria . An empirical probability distribution that is consistent for all E.coli promoters has been established . This information is combined with the results from NNPP2.2 to create a new technique called TLS-NNPP which improves the specificity of promoter prediction . The technique is shown to be effective using E-Coli DNA sequences, however it is applicable to any organism for which a set of promoters has been experimentally defined . AVAILABILITY: The data used in this project and the prediction results for the tested sequences can be obtained from http://www.uow.edu.au/yanxia/E_Coli_paper/SBurden_Results.xls. Mutat Res, 2004 Oct 4, 554(1-2), 95 - 109 Genetic recombination destabilizes (CTG)n.(CAG)n repeats in E . coli; Hashem VI et al.; The expansion of trinucleotide repeats has been implicated in 17 neurological diseases to date . Factors leading to the instability of trinucleotide repeat sequences have thus been an area of intense interest . Certain genes involved in mismatch repair, recombination, nucleotide excision repair, and replication influence the instability of trinucleotide repeats in both Escherichia coli and yeast . Using a genetic assay for repeat deletion in E . coli, the effect of mutations in the recA, recB, and lexA genes on the rate of deletion of (CTG)n.(CAG)n repeats of varying lengths were examined . The results indicate that mutations in recA and recB, which decrease the rate of recombination, had a stabilizing effect on (CAG)n.(CTG)n repeats decreasing the high rates of deletion seen in recombination proficient cells . Thus, recombination proficiency correlates with high rates of genetic instability in triplet repeats . Induction of the SOS system, however, did not appear to play a significant role in repeat instability, nor did the presence of triplet repeats in cells turn on the SOS response . A model is suggested where deletion during exponential growth may result from attempts to restart replication when paused at triplet repeats. Pediatr Blood Cancer, 2005 Jan, 44(1), 77 - 84 Neuroblastoma-specific cytotoxicity mediated by the Mash1-promoter and E . coli purine nucleoside phosphorylase; Arvidsson Y et al.; BACKGROUND: Neuroblastoma is derived from cells of neural crest origin and often expresses the transcription factor human achaete-scute homolog 1 (HASH1) . The aim of this study was to selectively kill neuroblastoma cells by expressing the suicide gene E . coli purine nucleoside phosphorylase (PNP) under the control of the Mash1 promoter, the murine homolog of HASH1 . PROCEDURE: The E . coli PNP gene regulated by the Mash1 promoter was cloned into an expression vector and transfected into neuroblastoma and non-neuroblastoma cell lines . After addition of the prodrug M2-fluoroadenine 9-beta-D-arabinofuranoside (F-araA) the cell-specific toxicity was examined . To optimize the cell specific activity, different sizes of the Mash1 promoter were analyzed in neuroblastoma cell lines and compared with the activity in non-neuroblastoma cells . RESULTS: Estimated as the percentages of CMV enhancer-promoter, the activity was significantly higher in the neuroblastoma cells, ranging from 17 to 58% when the shortest and the most active promoter was measured . The non-neuroblastoma cells yielded only 1-6% of the CMV promoter activity . When the shortest Mash1 promoter was combined with the E . coli PNP gene the cytotoxicity was 65% in the neuroblastoma cells with low cell death in the non-neuroblastoma cell lines, relative to the cytotoxicity where the E.coli PNP gene was regulated by the strong but non-specific CMV enhancer-promoter . CONCLUSIONS: We show here that the Mash1 promoter regulating the PNP gene confers a cell-type selective toxicity in neuroblastoma cell lines . These results indicate the feasibility to use the Mash1 promoter for regulating E.coli PNP expression in gene-directed enzyme prodrug therapy (GDEPT) of neuroblastoma . (c) 2004 Wiley-Liss, Inc. Zhonghua Yi Xue Za Zhi, 2004 Aug 2, 84(15), 1294 - 8 {Experimental study on phenotypic conversion of clinical chloromycetin-resistant strains of E . coli to drug-sensitive strains by using EGS technique in vitro}; Gao MY et al.; OBJECTIVE: To explore the possibility of phenotypic conversion of clinical chloromycetin (Cm)-resistant isolates of E.coli to drug-sensitive ones with external guide sequences (EGS) in vitro . METHODS: Recombinant EGS plasmids directed against Cm acetyl transferase (cat) and containing kanamycin (Km) drug-resistance gene and control plasmids only containing kanamycin-resistance gene without EGS were constructed . By using CaCl(2) method, the recombinant plasmids were introduced into the clinically isolated Cm-resistant E.coli strains . Extraction of plasmids and PCR were applied to identify the EGS positive clones; The growth rate in liquid broth culture of Cm-resistant bacteria after EGS containing plasmid transformation was determined by spectrophotometer A(600) . Drug sensitivity was tested in solid culture by using KB method . RESULTS: Transformation studies were carried out on 16 clinically isolated Cm-resistant E.coli strains with pEGFP-C1-EGS + cat1 + cat2 recombinant plasmid . Transformants were screened on LB-agar plates containing Km after transformation using EGS . In 4 tested strains of them, transformants with specific EGS plasmid showed growth inhibition when grown in liquid broth culture containing 100 approximately 200 micro g/ml of Cm . They were sensitive to Cm on LB-agar plates containing 100 approximately 200 micro g/ml of Cm in drug-sensitivity test . Extraction of plasmids showed the existence of EGS bands . PCR amplified products of EGS . The above facts indicated that the 4 strains out of the 16 clinical isolates had been converted to drug-sensitive phenotype, and Cm-resistant clinically isolated E . coli resumed sensitivity to Cm . CONCLUSION: EGS has the capability of converting the phenotype of clinical drug-resistant isolates to drug sensitivity. J Gene Med, 2004 Oct, 6(10), 1092 - 101 Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E . coli beta-glucuronidase; Smahel M et al.; BACKGROUND: Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity . In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb-binding site of the HPV16 E7 protein . METHODS AND RESULTS: To increase E7GGG immunogenicity we constructed fusion genes of E . coli beta-glucuronidase (GUS) with one or three copies of E7GGG . Furthermore, a similar construct was prepared with partial E7GGG (E7GGGp, 41 amino acids from the N-terminus) . The expression of the fusion genes was examined in human 293T cells . Quantification of GUS activity and the amount of E7 antigen showed substantially reduced GUS activity of fusion proteins with complete E7GGG that was mainly caused by decrease of their steady-state level in comparison with GUS or E7GGGpGUS . Still, the steady-state level of E7GGG.GUS was about 20-fold higher than that of the E7GGG protein . The immunogenicity of the fusion genes with complete E7GGG was tested by DNA immunisation of C57BL/6 mice with a gene gun . TC-1 cells and their clone TC-1/A9 with down-regulated MHC class I expression were subcutaneously (s.c.) inoculated to induce tumour formation . All mice were protected against challenge with TC-1 cells and most animals remained tumour-free in therapeutic-immunisation experiments with these cells, in contrast to immunisation with unfused E7GGG and the fusion with the lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1) . Significant protection was also recorded against TC-1/A9 cells . Both tetramer staining and ELISPOT assay showed substantially higher activation of E7-specific CD8+ lymphocytes in comparison with E7GGG and Sig/E7GGG/LAMP-1 . Deletion of 231 bp in the GUS gene eliminated enzymatic activity, but did not influence the immunogenicity of the E7GGG.GUS gene . CONCLUSIONS: The findings demonstrate the superior immunisation efficacy of the fusion genes of E7GGG with GUS when compared with E7GGG and Sig/E7GGG/LAMP-1 . The E7GGG.GUS-based DNA vaccine might also be efficient against human tumour cells with reduced MHC class I expression. Rapid Commun Mass Spectrom, 2004, 18(19), 2201 - 7 A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E . coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer; Wu SL et al.; A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E . coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer . In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages . The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa) . The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell . The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run . Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified . The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH . The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies . On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration) . The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method . Mol Cell, 2004 Sep 24, 15(6), 965 - 75 Exchange of DNA base pairs that coincides with recognition of homology promoted by E . coli RecA protein; Folta-Stogniew E et al.; The unresolved mechanism by which a single strand of DNA recognizes homology in duplex DNA is central to understanding genetic recombination and repair of double-strand breaks . Using stopped-flow fluorescence we monitored strand exchange catalyzed by E . coli RecA protein, measuring simultaneously the rate of exchange of A:T base pairs and the rates of formation and dissociation of the three-stranded intermediates called synaptic complexes . The rate of exchange of A:T base pairs was indistinguishable from the rate of formation of synaptic complexes, whereas the rate of displacement of a single strand from complexes was five to ten times slower . This physical evidence shows that a subset of bases exchanges at a rate that is fast enough to account for recognition of homology . Together, several studies suggest that a mechanism governed by the dynamic structure of DNA and catalyzed by diverse enzymes underlies both recognition of homology and initiation of strand exchange . J Biol Chem, 2004 Dec 3, 279(49), 51156 - 62 Epub 2004 Dec 3. The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E . coli replication origin; Felczak MM et al.; Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process . Structure-function studies indicate that the replication initiator comprises four domains . Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J . P., Pirruccello, M . M., and Berger, J . M . (2002) EMBO J . 21, 4763-4773) . Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E . coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif . All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo . Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication . Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC . Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase . Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Sep, 20(5), 563 - 7 {Expression of antibody Fab against platelet IIb/IIIa in E.coli and characterization of its biological activity}; Yang YC et al.; AIM: To develop a Fab antibody against platelet GPIIb/IIIa . METHODS: A mouse anti-human GPIIb/IIIa monoclonal antibody (mAb) P140 was selected by the indirect immunofluorescence assay (IFA) and platelet aggregation inhibition test . The Fd and light chain genes were cloned from the hybridoma cells secreting the mAb P140 . The genes of P140 Fab were inserted into plasmid p3MH to construct recombinant expression plasmid p3MH/P140kappa-Fd . After digestion with the restriction enzyme, the recombinant plasmid was transformed into E.coli XLI-Blue . The expressed product was purified by TALON metal affinity resin . Purified P140 Fab was characterized by SDS-PAGE, ELISA, Western blot and platelet aggregation inhibition test . RESULTS: SDS-PAGE analysis showed that relative molecular mass (M(r)) of P140 Fab was 47x10(3) . The results of ELISA, Western blot and platelet aggragation inhibition test indicated that P140 Fab could specifically bind to platelet and inhibit platelet aggragation in dose-dependent manner . The mean value of IC(50) was 16.85 mg/L . CONCLUSION: A soluble anti-platelet GPIIb/IIIa antibody P140 Fab was prepared successfully, which lays the foundation for further clinical application. FEMS Microbiol Lett, 2004 Sep 15, 238(2), 321 - 32 Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E . coli strain from a diseased animal; Dezfulian H et al.; Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E . coli fimbrial adhesins . This strain produces two fimbriae: F165(1) and F165(2) . F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon . Data from this study suggest that these two operons are part of two PAIs . PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E . coli chromosome . PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes . PAI II(4787) is found between the proA and yagU at 6 min of the E . coli chromosome. Biochimie, 2004 Jun, 86(6), 403 - 9 Metal-ion induced conformational changes in alkaline phosphatase from E . coli assessed by limited proteolysis; Bucevic-Popovic V et al.; Alkaline phosphatase (AP) displays significant structural changes during metal-ion binding, supporting cooperative interactions between the subunits of the dimeric enzyme . Here, we present data on the dynamic properties of AP from E . coli, and characterize the structural changes that accompany variations in metal-ion content, combining limited proteolysis and MALDI-TOF mass spectrometry . Limited proteolysis revealed an internal cleavage site at Arg-293, reflecting a position of conformational flexibility supporting subunit communication essential for catalysis . A specific shielding of a region distant from the metal-binding site has been demonstrated, implying transmission of conformational changes, induced by metal-ion binding to the adjacent subunit, across the subunit interface. Biotechnol Bioeng, 2004 Sep 5, 87(5), 641 - 7 Extreme scale-down of expanded bed adsorption: Purification of an antibody fragment directly from recombinant E . coli culture; Willoughby N et al.; Scale-down is a methodology that combines the use of very small volumes of process fluid in dedicated devices to predict accurately the behaviour of process-scale biotechnological unit operations and for the production of comparable material for use in further devices which, taken together, facilitate the mimic of a complete full-scale process . This article provides the rationale behind the development of a small-scale mimic and demonstrates the use of a highly scaled-down expanded bed to predict hydrodynamic, kinetic, and adsorptive performance using less than 5-mL sample volumes . Data acquired on a specially developed 1.9 mm ID column was compared with that obtained in a standard 25 mm ID column . A homogenised E . coli system expressing an antibody fragment (F(ab)) adsorbed onto an rProtein A matrix was used to characterise the full adsorptive performance . Breakthrough curve studies using BSA in buffer were used to characterise binding kinetics . Performance at the two scales was comparable both in terms of expansion, axial dispersion, binding isotherms, and elution behaviour of the antibody fragment . The eluted F(ab) material was further purified by ion exchange chromatography to demonstrate the similarity between the profile of the product material obtained at both scales . The high level of scale-down (approximately 200-fold) provides for rapid process evaluation early in development, where material is at a premium and where a fast appreciation of the likely merits of one process strategy will lead to greater confidence in process selection and more robust flowsheets. Shi Yan Sheng Wu Xue Bao, 2002 Jun, 35(2), 89 - 97 {Expression of monoclonal nonspecific suppressor factor beta in E . coli, preparation of its antibodies and its tissue orientation in the mouse endometrium}; Huang ZP et al.; In order to explore the role of MNSFbeta in the process of implantaion, MNSFbeta and its antibodies are required . The expression plasmid pBV220/MNSFbeta-hCGbeta was constructed, and then transferred into E . coli to express the fusion protein MNSFbeta-hCGbeta . The anti-hCGbeta antibody was used to identify the fusion protein . The result demonstrated that MNSFbeta-hCGbeta was expressed correctly and its molecular weight was consistent with the anticipated one . Finally the expression product MNSFbeta-hCGbeta was preliminarily purified and used to immunize Balb/C mouse to generate the antibodies . In the meantime, the expression plasmid pGEX-4T-2/MNSFbeta was also constructed and transferred into E . coli to express the fusion protein GST-MNSFbeta . GST-MNSFbeta was purified and used to stimulate the immunized mouse before the preparation of hybridomas cells . The prepared polyclonal and monoclonal antibodies against MNSFbeta were checked and measured by fusion protein GST-MNSFbeta . The prepared polyclonal antibody was then used to perform the immunohistochemistry analysis . The result suggested that the level of MNSFbeta in interimplantation sites was significantly higher as compared with implantation sites in the mouse uterine on Day 4.5 of pregnancy. Genome Res, 2004 Sep, 14(9), 1797 - 805 Genome-scale in silico models of E . coli have multiple equivalent phenotypic states: assessment of correlated reaction subsets that comprise network states; Reed JL et al.; The constraint-based analysis of genome-scale metabolic and regulatory networks has been successful in predicting phenotypes and useful for analyzing high-throughput data sets . Within this modeling framework, linear optimization has been used to study genome-scale metabolic models, resulting in the enumeration of single optimal solutions describing the best use of the network to support growth . Here mixed-integer linear programming was used to calculate and study a subset of the alternate optimal solutions for a genome-scale metabolic model of Escherichia coli (iJR904) under a wide variety of environmental conditions . Analysis of the calculated sets of optimal solutions found that: (1) only a small subset of reactions in the network have variable fluxes across optima; (2) sets of reactions that are always used together in optimal solutions, correlated reaction sets, showed moderate agreement with the currently known transcriptional regulatory structure in E . coli and available expression data, and (3) reactions that are used under certain environmental conditions can provide clues about network regulatory needs . In addition, calculation of suboptimal flux distributions, using flux variability analysis, identified reactions which are used under significantly more environmental conditions suboptimally than optimally . Together these results demonstrate the utilization of reactions in genome-scale models under a variety of different growth conditions. Eksp Klin Farmakol, 2004 May-Jun, 67(3), 56 - 8 {The functional state of thymus cells under prolonged combined action of phenobarbital and E . coli lipopolysaccharide}; Zhitkevich TI et al.; The effect of separate and combined 10-day treatment with phenobarbital (PB) in a single daily dose of 35 mg/kg and E . coli lipopolysaccharide (LPS) in a dose of 25 microg/kg was studied in white rats . The results were evaluated by the rate of DNA synthesis in the culture of T mitogen stimulated thymocytes, the activity of glucose-6-phosphate dehydrogenase (G-6-PDG) in the thymus, and the activity of acetylcholine esterase (ACE) and the content of catecholamines (CA) in the nerve fibers of this organ . It was established that the 10-day treatment with PB inhibits the proliferative activity in concanavalin A stimulated lymphocytes of rat thymus, decreases the ACE activity, and increases the CA content in the nerve fibers . The repeated injections of E . coli LPS on the background of PB led to more pronounced negative changes in the functional activity of lymphoid cells in the thymus, manifested by a decrease in the G-6-PDG activity and the rate of DNA synthesis in the thymus and by an increase in the level of biogenic amines in the nervous fibers of this organ. J Mol Model (Online) . 2004 Aug 27; {Epub ahead of print} Modeling the E . coli 4-hydroxybenzoic acid oligoprenyltransferase ( ubiA transferase) and characterization of potential active sites; Brauer L et al.; 4-Hydroxybenzoate oligoprenyltransferase of E . coli, encoded in the gene ubiA, is an important key enzyme in the biosynthetic pathway to ubiquinone . It catalyzes the prenylation of 4-hydroxybenzoic acid in position 3 using an oligoprenyl diphosphate as a second substrate . Up to now, no X-ray structure of this oligoprenyltransferase or any structurally related enzyme is known . Knowledge of the tertiary structure and possible active sites is, however, essential for understanding the catalysis mechanism and the substrate specificity.With homology modeling techniques, secondary structure prediction tools, molecular dynamics simulations, and energy optimizations, a model with two putative active sites could be created and refined . One active site selected to be the most likely one for the docking of oligoprenyl diphosphate and 4-hydroxybenzoic acid is located near the N-terminus of the enzyme . It is widely accepted that residues forming an active site are usually evolutionary conserved within a family of enzymes . Multiple alignments of a multitude of related proteins clearly showed 100% conservation of the amino acid residues that form the first putative active site and therefore strongly support this hypothesis . However, an additional highly conserved region in the amino acid sequence of the ubiA enzyme could be detected, which also can be considered a putative (or rudimentary) active site . This site is characterized by a high sequence similarity to the aforementioned site and may give some hints regarding the evolutionary origin of the ubiA enzyme.Semiempirical quantum mechanical PM3 calculations have been performed to investigate the thermodynamics and kinetics of the catalysis mechanism . These results suggest a near S(N)1 mechanism for the cleavage of the diphosphate ion from the isoprenyl unit . The 4-hydroxybenzoic acid interestingly appears not to be activated as benzoate anion but rather as phenolate anion to allow attack of the isoprenyl cation to the phenolate, which appeared to be the rate limiting step of the whole process according to our quantum chemical calculations . Our models are a basis for developing inhibitors of this enzyme, which is crucial for bacterial aerobic metabolism.FIGURE Structure of the model of ubiA oligoprenyltransferase derived from the photosynthetic reaction center (1PRC) . Putative active amino acid residues and substrates are shown as capped sticks to describe their location and geometry in the putative active sites . The violet spheres identify Mg(2+). J Mol Biol, 2004 Sep 10, 342(2), 489 - 502 Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme; Sulzenbacher G et al.; In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge . The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols . The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3) . YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain . The crystals contain two dimers in the asymmetric unit . While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites . The active site contains a Zn atom, as verified by EXAFS on the crystals . The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6 . Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2) . These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions. J Mol Biol, 2004 Sep 10, 342(2), 467 - 78 Geometric and dynamic requirements for DNA looping, wrapping and unwrapping in the activation of E.coli glnAp2 transcription by NtrC; Lilja AE et al.; Transcriptional activation by the E.coli NtrC protein can occur via DNA looping between a DNA-bound activator and the target sigma(54) RNA polymerase . NtrC forms an octamer on DNA that is capable of binding two DNA molecules . Its ATPase activity is required for open complex formation . Geometric requirements for activation were assessed using a library of DNA bending sequences created by random ligation of A-tract oligonucleotides, as well as several designed sequences . Thirty random or designed sequences with a variety of DNA lengths and bending geometries were cloned in plasmids, and the library was used to replace the spacer between the NtrC binding sites and the core glnAp2 promoter . The activity of each promoter construct under nitrogen limitation was determined in vivo, in a lambda phage lacZ reporter system integrated as a single-copy lysogen to avoid titrating NtrC or polymerase . A wide variety of bending geometries was found to support a similar level of transcriptional activation ( approximately 3-4-fold) . Computer modeling of the DNA trajectories suggests that the most inactive promoters have short spacer DNA and the NtrC sites on the opposite side of the helix as the wild-type sites; otherwise, the loop can form effectively . Flexibility and multivalency of the NtrC-Esigma(54) interaction apparently provides substantial independence from DNA stiffness constraints, and in general activation requires less efficient looping than repression . However, none of the random templates were as active as wild-type promoter . Subsidiary activator binding sites in the wild-type were found to be required for full activity, but, surprisingly, these sites could not be functionally replaced by strong binding sites . This suggests that one or more protomers in the NtrC octamer must form and then release contacts with DNA in order to complete the ATPase cycle and act as an AAA(+) activator of the Esigma(54) . This dynamic DNA wrapping around the NtrC octamer is proposed to be necessary for efficient activation, and the wrapping may also reduce adventitious activation of other promoters. Water Sci Technol, 2004, 50(1), 233 - 7 Development of procedures for rapid detection of E . coli O157:H7 from source and finished water samples; Bukhari Z et al.; Enterohaemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent for haemolytic uraemic syndrome, has become a significant health concern, due to an increasing number of cases . The focus of this project was rapid (<8 h) detection of E . coli O157:H7 from source and finished waters samples, either directly or indirectly, by dovetailing the procedures with existing total coliform procedures . Evaluation of four immunological lateral diffusion assays determined a detection range between 1 x 10(4) and 1 x 10(6) CFU, with the Reveal E . coli O157:H7 Test System (Neogen) being the most sensitive for detecting E . coli O157:H7 . Evaluation of the BAX System for molecular detection determined that as few as 10 CFU could be reproducibly detected . Coupling either of these detection procedures with organism propagation using Tryptic Soy Broth (TSB) enabled sufficient quantities of E . coli O157:H7, such that the Reveal and BAX detection methods could be used with the 8 h time frame . Examination of matrix effects on the overall procedure indicated little impact on method performance. Methods Mol Biol, 2004, 278, 1 - 16 Screening and optimizing protein production in E . coli; Hewitt L et al.; Significant improvements in the technologies used for protein production have been driven by impending genome-scale proteomics projects . These initiatives have favored Escherichia coli-based expression systems, which allow rapid cloning and expression of proteins at low cost . The range of commercially available molecular biology kits, vectors, affinity tags, and host cell lines have increased dramatically in recent years . For the structural biology community, where protein production is often a rate-limiting step, these developments have made the process of producing and purifying large amounts of protein for structural studies simpler and faster . The large-scale automated screening approaches for optimizing protein production employed by structural genomics initiatives can be adapted to a more practical targeted approach appropriate for individual structural biology groups . This chapter describes simple, rapid screening methods for testing optimal vector/host combinations using a 96-well format. Hepatobiliary Pancreat Dis Int, 2004 Aug, 3(3), 417 - 22 A rabbit model of non-cirrhotic portal hypertension by repeated injections of E.coli through indwelling cannulation of the gastrosplenic vein; Omanwar S et al.; BACKGROUND: Non-cirrhotic portal hypertension is a common cause of portal hypertension in developing countries . To understand its etiopathogenesis we developed an animal model by repeated portal endotoxemia induced through the gastrosplenic vein . METHODS: Twenty-nine rabbits (1.5-2.0 kg) were divided into control (group I, n=13) and experimental (group II, n=16) groups . Heat killed E.coli were injected through an indwelling cannula into the gastrosplenic vein in pre-sensitized animals . The animals were sacrificed at 1, 3 and 6 months . RESULTS: The mean portal pressure in group II animals was significantly (P<0.05) higher than in group I at 1 (17.5+/-3.4 vs 10.4+/-2.2 mmHg), 3 (17.8+/-1.3 vs 7.2+/-3.6 mm Hg), and 6 (19.8+/-3.1 vs 10.3+/-4.8 mmHg) months . Similarly, the mean splenic weight in group II was significantly greater than in group I (P<0.05) . Histopathologically, the spleen showed medullary congestion, hemosidrin-laden macrophages and mild fibrosis . Histologically, the liver had normal parenchyma with mild portal lymphocytic infiltrates and kupffer cell hyperplasia . No significant anomalies were detected by liver function tests . CONCLUSIONS: The rabbit model showed significant splenomegaly with a persistent increase in portal pressure and mild fibrosis without hepatic parenchymal injury, quite akin to non-cirrhotic portal fibrosis as seen in humans . Recurrent intra-abdominal infection may play an important role in the pathogenesis of non-cirrhotic portal fibrosis. J Mol Biol, 2004 Sep 3, 342(1), 229 - 45 Exploring the structural dynamics of the E.coli chaperonin GroEL using translation-libration-screw crystallographic refinement of intermediate states; Chaudhry C et al.; Large rigid-body domain movements are critical to GroEL-mediated protein folding, especially apical domain elevation and twist associated with the formation of a folding chamber upon binding ATP and co-chaperonin GroES . Here, we have modeled the anisotropic displacements of GroEL domains from various crystallized states, unliganded GroEL, ATPgammaS-bound, ADP-AlFx/GroES-bound, and ADP/GroES bound, using translation-libration-screw (TLS) analysis . Remarkably, the TLS results show that the inherent motions of unliganded GroEL, a polypeptide-accepting state, are biased along the transition pathway that leads to the folding-active state . In the ADP-AlFx/GroES-bound folding-active state the dynamic modes of the apical domains become reoriented and coupled to the motions of bound GroES . The ADP/GroES complex exhibits these same motions, but they are increased in magnitude, potentially reflecting the decreased stability of the complex after nucleotide hydrolysis . Our results have allowed the visualization of the anisotropic molecular motions that link the static conformations previously observed by X-ray crystallography . Application of the same analyses to other macromolecules where rigid body motions occur may give insight into the large scale dynamics critical for function and thus has the potential to extend our fundamental understanding of molecular machines. Biochemistry (Mosc), 2004 Jul, 69(7), 776 - 81 Recombinant Destabilase-Lysozyme: Synthesis de novo in E . coli and Action Mechanism of the Enzyme Expressed in Spodoptera frugiperda; Zavalova LL et al.; Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families . In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli . Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial, leech, or yeast signal peptides (SP) were used . The use of a construct without signal peptide genes resulted in normal growth of the transformed cells . Transformation of E . coli cells with the constructs containing SP was accompanied by the disruption of the forming cells . The use of the expression vector pET-32 LTC-System for production of DL as a fusion protein with thioredoxin also resulted in normal cell growth . However, specific activity of DL isolated from such cells was significantly lower than that of enzyme purified from extracts of Spodoptera frugiperda cells, which were infected with the baculovirus vector carrying DL cDNA . It is shown that the action mechanism of invertebrate lysozyme does not differ from that of other families: recombinant DL from S . frugiperda extracts catalyzed cleavage of synthetic substrate, hexamer of N-acetylglucosamine, to di- and tetramers, which is typical for enzymatic function of other lysozyme families. Biochimie, 2004 Jul, 86(7), 495 - 500 Simultaneous binding of trigger factor and signal recognition particle to the E . coli ribosome; Raine A et al.; Signal recognition particle (SRP) and trigger factor (TF) both bind to ribosomal protein L23 at the peptide exit area on the 50S subunit of the E . coli ribosome . In this study, we have developed a spin-down assay and used it to estimate KD values and the corresponding enthalpies for the binding of radio-labelled SRP and TF to naked ribosomes and to ribosomes carrying a tetrapeptidyl-tRNA in the P site . At 20 degrees C, the KD value for TF binding is 2 microM and for SRP it is 170 nM for naked as well as for translating ribosomes . At 4 degrees C, the KD values for TF and SRP binding are 1.1 microM and 90 nM, respectively . Competition binding experiments reveal that SRP and TF bind simultaneously to the ribosome with little affinity interference, showing that the factors have separate binding sites on L23 . This makes an alternating binding mode for TF and SRP less plausible. Chembiochem, 2004 Aug 6, 5(8), 1088 - 94 Preliminary characterization of light harvesting in E . coli DNA photolyase; Henry AA et al.; E . coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA . For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm . Using the crystal structure as a guide, we mutated each protein residue that contacts the cofactor in an effort to identify the interactions responsible for this selective stabilization of the cofactor's excited state . Hydrogen bonding, packing, and electrostatic interactions were examined . Remarkably, a single residue, Glu109, appears to play an important, if not exclusive, role in inducing the observed red-shift . Thus, this protein, the simplest light-harvesting system known, appears to have evolved a remarkably simple mechanism to tune the photophysical properties of the antenna cofactor appropriately for biological function. Acta Crystallogr D Biol Crystallogr, 1997 Jan, 53(Pt 1), 129 - 30 Crystallization and preliminary X-ray studies of plastocyanin from Silene expressed in E . coli; Li C; Plastocyanin from Silene (SilPc) expressed in E . coli has been crystallized in a form suitable for X-ray diffraction analysis by a macroseeding method using ammonium sulfate as a precipitant in acetate buffer (pH = 5.5) . These crystals belong to the trigonal space group P3(1)21 or P3(2)21 with lattice parameters a = b = 76.6, c = 65.5 A, indicating an asymmetric unit containing two plastocya