Protein Expr Purif, 2005 Feb, 39(2), 283 - 287 Refolding and one-step purification of recombinant human ARA70 over-expressed in Escherichia coli; Singh VK et al.; Androgen receptor (AR)-associated coregulator 70 (ARA70) is a cytoplasmic protein that has been characterized to have the ability to induce AR transcriptional activity in response to androgens and anti-androgens in prostate cancer cells . AR has been shown to have an important role in the progression of prostate cancer and in normal male reproductive system development . To elucidate the molecular mechanisms and biological relevance of ARA70 to prostrate cancer using a variety of biochemical analyses, the cDNA encoding full length ARA70 was cloned into pET21b vector . Here, we report the refolding and one-step purification of ARA70 from inclusion bodies over-expressed in Escherichia coli . The protein was purified to homogeneity, yielding approximately 60mg ARA70 from 1L of terrific broth media . Refolding process of ARA70 was monitored using far-UV CD analysis. Lett Appl Microbiol, 2005, 40(1), 74 - 80 Effect of human milk on type 1 and P-fimbrial mRNA expression in intestinal Escherichia coli strains; Nowrouzian FL et al.; Abstract f.l . nowrouzian, h.-j . monstein, a.e . wold and i . adlerberth . 2004.Aims: Escherichia coli from breastfed infants express more type 1 fimbriae and less P fimbriae than E . coli from bottle-fed infants . In this study we investigated the effect of human milk on production of mRNA for fimA (type 1 fimbriae) and papC (P fimbriae) in E . coli . Methods and Results: Production of adhesin gene mRNA was estimated using a reverse transcriptase polymerase chain reaction in E . coli strains under different culture conditions . More type 1 fimbrial mRNA was produced after culture in human milk (P = 0.001) or Luria broth (P = 0.014) than after culture on agar, whereas P-fimbrial mRNA production was similar under all tested growth conditions . When cultured on agar, E . coli strains carrying both the fim and pap operons produced less type 1 and P-fimbrial mRNA than strains that had only the fim or pap operons, respectively (P = 0.03 and 0.056) . Significance and Impact of the Study: Environmental regulation of adhesin expression may be influenced by cross-talk between fimbrial operons. Yakugaku Zasshi, 2004 Dec, 124(12), 983 - 7 {Detection of bacteria contaminating in blood for transfusion}; Osanai T et al.; When Escherichia coli is cultured in nutrient broth at 1 x 10(3) cells/ml, 7.5 h are needed to detect cell growth as turbidity . The time to detect E . coli in this medium is reduced using the alamar blue method . Alamar blue is an oxidation-reduction indicator that changes color in response to cell growth . E . coli can grow in nutrient broth containing red blood cells, but the detection of E . coli based on turbidity is difficult because the red blood cells muddy the medium . As the change in color in the alamar blue method is not affected by red blood cells, the growth of E . coli contaminating red blood cells is detectable . These results suggest that a cell growth indicator such as alamar blue is useful for the detection of bacteria contaminating blood for transfusion. Proc Natl Acad Sci U S A, 2004 Dec 14, 101(50), 17486 - 91 Epub 2004 Nov 22. Subcellular distribution of enzyme I of the Escherichia coli phosphoenolpyruvate:glycose phosphotransferase system depends on growth conditions; Patel HV et al.; The phosphoenolpyruvate:glycose phosphotransferase system (PTS) participates in important functions in the bacterial cell, including the phosphorylation/uptake of PTS sugars . Enzyme I (EI), the first protein of the PTS complex, accepts the phosphoryl group from phosphoenolpyruvate, which is then transferred through a chain of proteins to the sugar . In these studies, a mutant GFP, enhanced yellow fluorescent protein (YFP), was linked to the N terminus of EI, giving Y-EI . Y-EI was active both in vitro (>/=90% compared with EI) and in vivo . Unexpectedly, the subcellular distribution of Y-EI varied significantly . Three types of fluorescence were observed: (i) diffuse (dispersed throughout the cell), (ii) punctate (concentrated in numerous discrete spots throughout the cell), and (iii) polar (at one or both ends of the cell) . Cells from dense colonies grown on agar plates with LB broth or synthetic (Neidhardt) medium showed primarily bipolar or punctate fluorescence . In liquid culture, under carefully defined carbon-limiting growth conditions {ribose (non-PTS), mannitol (PTS sugar), or dl-lactate}, cellular levels of enzymatically active Y-EI remain essentially constant for each carbon source, but fluorescence distribution depends on C source, cell density, growth phase, and apparently on "conditioned medium." Fluorescence was diffuse during exponential growth on LB or ribose/Neidhardt medium . On ribose they became punctate in the stationary phase, reverting to diffuse when more ribose was added . In LB, both Y-EI and a nonphosphorylatable mutant, H189Q-Y-EI, showed a diffuse fluorescence during growth, but, shortly after the addition of isopropyl beta-d-thiogalactopyranoside, Y-EI became bipolar; H189Q-Y-EI did not . The functions of EI sequestration remain to be determined. J Food Prot, 2004 Aug, 67(8), 1604 - 9 Rapid method for prediction of Escherichia coli numbers using an electronic sensor array and an artificial neural network; Siripatrawan U et al.; An electronic sensor array with 12 nonspecific metal oxide sensors was evaluated for its ability to monitor volatile compounds in super broth alone and in super broth inoculated with Escherichia coli (ATCC 25922) at 37 degrees C for 2 to 12 h . Using discriminant function analysis, it was possible to differentiate super broth alone from that containing E . coli when cell numbers were 10(5) CFU or more . There was a good agreement between the volatile profiles from the electronic sensor array and a gas chromatography-mass spectrometer method . The potential to predict the number of E . coli and the concentration of specific metabolic compounds was investigated using an artificial neural network (ANN) . The artificial neural network was composed of an input layer, one hidden layer, and an output layer, with a hyperbolic tangent sigmoidal transfer function in the hidden layer and a linear transfer function in the output layer . Good prediction was found as measured by a regression coefficient (R2 = 0.999) between actual and predicted data. J Food Prot, 2004 Aug, 67(8), 1597 - 603 Solid-phase microextraction, gas chromatography, and mass spectrometry coupled with discriminant factor analysis and multilayer perceptron neural network for detection of Escherichia coli; Siripatrawan U et al.; This study was performed to investigate the ability of using discriminant factor analysis (DFA) and an artificial neural network (ANN) to identify and quantify the number of Escherichia coli (ATCC 25922) in nutrient media from data generated by analysis of E . coli volatile metabolic compounds using solid-phase microextraction (SPME) coupled with gas chromatography (GC) and mass spectrometry (MS) . E . coli was grown in super broth and incubated at 37 degrees C for 2 to 12 h . Numbers of E . coli were followed using a colony counting method . An SPME device was used to collect the volatiles from the headspace above the samples, and the volatiles were identified using GC-MS . DFA was used to classify the samples from different incubation times . From DFA, it was possible to differentiate super broth from media containing E . coli when cell numbers were 10(5) CFU or more . The potential to predict the number of E . coli from the SPME-GC-MS data was investigated using a multilayer perceptron (MLP) neural network with back propagation training . The MLP comprised an input layer, one hidden layer, and an output layer, with a hyperbolic tangent sigmoidal transfer function in the hidden layer and a linear transfer function in the output layer . Good prediction was found as measured by a regression coefficient (R2 = 0.996) between actual and predicted data. J Basic Microbiol, 2004, 44(4), 296 - 304 Lectin-binding epitopes at the surface of Escherichia coli K-12: examination by electron microscopy, with special reference to the presence of a colanic acid-like polymer; Stoitsova S et al.; The presence and distribution of lectin-binding epitopes at the surface of Escherichia coli K-12, strain W1655, was studied by electron microscopy after lectin-gold labeling and negative staining . A comparison was made between the lectin-binding capacity of cells cultivated at 20 degrees C and 37 degrees C (in broth or on agar) . A variety of pre-treatment protocols were applied prior to labeling . The gold-conjugated lectins used were wheat germ agglutinin (WGA), soybean agglutinin (SBA) and Ulex europaeus lectin (UEA-I) . For all culture conditions, the bacteria had moderate exposure of WGA-binding sites, and this was not changed after pre-treatment . Cells cultivated at 37 degrees C had exposed SBA- and UEA-I-binding epitopes apparently associated with the cell surface . These significantly increased in number after boiling the cells for 10 min . With bacteria cultivated at 20 degrees C these two lectins recognized sites situated on exopolysaccharide filaments . Affino dot-blot experiments with isolated polysaccharides of the strain identified the K-12 lipooligosaccharide as the source of WGA-binding epitopes, and the exopolysaccharide, colanic acid (CA) as the source of SBA- and UEA-I-binding sites . The interaction with these two lectins of bacteria cultivated at 37 degrees C could be due to altered translocation of CA from the cytoplasm to the environment . This suggestion was supported by the demonstration by electron microscopy of SBA and UEA-I binding at the surface of hot phenol-water extracted cell walls . Appl Environ Microbiol, 2004 Jul, 70(7), 3893 - 7 Isolation and characterization of Micromonospora phage PhiHAU8 and development into a phasmid; Li X et al.; PhiHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp . strains 40027 and A-M-01, was isolated . The PhiHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca . 42.5 kb) with double-stranded DNA and cohesive ends . PhiHAU8 was most stable at 4 degrees C in Difco nutrient broth within a pH range of 6 to 12 . PhiHAU8 plaque formation on Micromonospora sp . strain 40027 was optimal with 32 mM Ca(2+) and 30 mM Mg(2+) . A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a lambda cosmid vector in Escherichia coli and as a phage in Micromonospora sp . strain 40027 was constructed . Pulsed-field gel electrophoresis of AseI-digested total DNA showed that PhiHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp . strain 40027. Peptides, 2004 May, 25(5), 785 - 92 Bacterial expression and purification of biologically active human TFF3; Fang M et al.; A glutathione S-transferase (GST) fusion protein expression system for the production and purification of recombinant human trefoil factor family-domain peptide 3 (hTFF3) was established . The hTFF3 gene, prepared by PCR, was cloned into a pBluescript KS(+) plasmid, and inserted into a pGEX-4T-1 GST fusion vector . The GST-hTFF3 fusion protein was expressed in Escherichia coli, and hTFF3 was purified with Glutathione Sepharose 4B affinity chromatography, yielding about 3-4 mg of pure hTFF3 in one liter of culture broth . The biological activity of purified hTFF3 was tested in two previously reported rat gastric ulcer models . Oral administration of recombinant hTFF3 has a dose dependent protective effect against ethanol-induced or pylorus ligation-induced gastric mucosa injury in rat, which indicates that our recombinant hTFF3 is biologically active. Bioprocess Biosyst Eng, 2004 Apr, 26(3), 147 - 50 High cell density fed-batch cultivation of Escherichia coli using exponential feeding combined with pH-stat; Kim BS et al.; A new feeding strategy in fed-batch culture, exponential feeding combined with pH-stat is suggested to avoid the accumulation of substrate in culture broth . Exponential feeding was stopped whenever a predetermined amount of limiting substrate was supplied and then pH change was observed . When pH rose above an upper limit due to the depletion of substrate, feeding was restarted . With this feeding strategy, recombinant Escherichia coli could be grown to 101 g/l by controlling the specific growth rate at 0.1 h(-1). Infect Immun, 2004 Jun, 72(6), 3218 - 27 Comparative analysis of EspF from enteropathogenic and enterohemorrhagic Escherichia coli in alteration of epithelial barrier function; Viswanathan VK et al.; Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E . coli (EHEC) are related intestinal pathogens that harbor highly similar pathogenicity islands known as the locus of enterocyte effacement (LEE) . Despite their genetic similarity, these two pathogens disrupt epithelial tight junction barrier function with distinct kinetics . EHEC-induced reduction in transepithelial electrical resistance (TER), a measure of barrier function disruption, is significantly slower and more modest in comparison to that induced by EPEC . The variation in bacterial adherence only partially accounted for these differences . The LEE-encoded effector protein EspF has been shown to be critical for EPEC-induced alterations in TER . EspF from both EPEC and EHEC is expressed and secreted upon growth in tissue culture medium . The mutation of EHEC cesF suggested that the optimal expression and secretion of EHEC EspF required its chaperone CesF, as has been shown for EPEC . In contrast to EPEC espF and cesF, mutation of the corresponding EHEC homologs did not dramatically alter the decrease in TER . These differences could possibly be explained by the presence of additional espF-like sequences (designated U- and M-espF, where the letter designations refer to the specific cryptic prophage sequences on the EHEC chromosome closest to the respective genes) in EHEC . Reverse transcription-PCR analyses revealed coordinate regulation of EHEC U-espF and the LEE-encoded espF, with enhanced expression in bacteria grown in Dulbecco-Vogt modified Eagle's medium compared to bacteria grown in Luria broth . Both EHEC espF and U-espF complemented an EPEC espF deletion strain for barrier function alteration . The overexpression of U-espF, but not espF, in wild-type EHEC potentiated the TER response . These studies reveal further similarities and differences in the pathogenesis of EPEC and EHEC. Water Res, 2004 May, 38(9), 2367 - 73 Recovery of Escherichia coli in fresh water fish, Jenynsia multidentata and Bryconamericus iheringi; Guzman MC et al.; Escherichia coli concentration was determined in digestive tract and muscle of Jenynsia multidentata and Bryconamericus iheringi through bioassays . Field experiments were also conducted with J . multidentata collected in the Suquia River, Cordoba, Argentina . E . coli was quantified by the most probable number, using lauryl sulphate tryptose broth with 4-methylumbelliferyl-beta-D-glucuronide . For bioassays, E . coli concentrations 10(2), 10(3), 10(4), 10(5), 10(6)CFU/ml were introduced in aquarium water . E . coli was recovered from the digestive tracts of J . multidentata and B . iheringi in all the concentrations assayed . Bacterial critical load in water for the recovery of bacteria from muscle, was 10(3)CFU/ml for both species . The regression analysis between E . coli loads in water and those found in digestive tract and muscle showed a positive linear relationship for J . multidentata and B . iheringi . The same relation was observed between the concentration of bacteria in digestive tract and muscle in both species . In field experiments, E . coli was recovered from digestive tract and muscle of J . multidentata . The presence of E . coli in the studied fish suggests that they can carry bacteria to non-polluted waters . However, further studies are necessary to evaluate its significance for public and environmental health. Appl Biochem Biotechnol, 2004 Spring, 113-116, 453 - 68 Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins; Penna TC et al.; To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4 degrees C prior to incubation, agitation speed, isopropyl-beta-D-thiogalactopyranoside {IPTG} concentration, and induction time) . The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method . To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfate polyacrylamide gel . With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample . TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass . Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37 degrees C, 24-h incubation). Oral Microbiol Immunol, 2004 Feb, 19(1), 16 - 25 Two epithelial cell invasion-related loci of the oral pathogen Actinobacillus actinomycetemcomitans; Li L et al.; Two invasion-related loci, apiA and the two-gene operon apiBC, were isolated from the oral pathogen Actinobacillus actinomycetemcomitans UT32 . apiA encodes a 32.5 kDa protein that migrates on SDS-PAGE as a 101 kDa protein as detected by Western blot analysis or silver staining of an outer membrane-enriched fraction of Escherichia coli transformants . E . coli expressing ApiA have a different phenotype than the host vector, in broth and on solid media, and a colony morphology that resembles that of fresh A . actinomycetemcomitans isolates . These E . coli transformants bound to chicken collagen type II, human collagen type II, III, V and fibronectin . apiB and apiC encode proteins of 130.1 and 70.6 kDa, respectively . ApiBC conferred on E . coli a slightly enhanced ability to bind to collagen type III . ApiA- and ApiB-deficient mutants were constructed in A . actinomycetemcomitans . The ApiB-mutant had 4-fold diminished invasion of KB cells; the ApiA-mutant had increased invasion . Both loci were found in all A . actinomycetemcomitans strains, although polymorphism was detected only for apiBC . The deduced sequences of these invasion-related proteins are homologous to members of the YadA adhesin/invasin family. J Bacteriol, 2003 Dec, 185(24), 7044 - 52 The growth advantage in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment; Farrell MJ et al.; Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype . A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity . However, it is important to note that during long-term batch culture, environmental conditions are in flux . To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture . To determine what role environmental conditions play in rendering relative fitness advantages to E . coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated . In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions . Similar results were found using other media . rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy . In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage . In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture . We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent. Int J Food Microbiol, 2003 Nov 15, 88(1), 55 - 61 Behaviour of log-phase Escherichia coli at temperatures near the minimum for growth; Jones T et al.; The behaviour of cold-adapted, log-phase Escherichia coli in broth cultures incubated at temperatures between 7 and 15 degrees C was examined by determinations of numbers of colonies recovered on plate count agar (PCA); absorbance at 600 nm (A600); cell lengths from photomicrographs; and cell size distributions by flow cytometry . Cultures incubated between 7 and 10 degrees C were evaluated for 8 days or until A600 values approached 1.0 . Cultures incubated at > or =12 degrees C were subcultured to maintain them in the log phase for up to 8 days . Numbers of colonies recovered declined when cultures were incubated at 7 degrees C, but increased when cultures were incubated at higher temperatures . However, A600 values increased during incubation at all temperatures . The mean lengths of cells doubled during incubation at 7 degrees C for 8 days, but remained constant during incubation at 10 degrees C for 1.25 days . Forward angle light scatter (FALS) measurements obtained by flow cytometry indicated that the mean length of cells increased at < or = 8 degrees C, but not at 10 degrees C . A reference value at the 90th percentile of FALS measurements on day 0 was used to determine changes in the distribution of the lengths of cells . About 80% or 17% of the cells were above the reference value after 5 days of incubation at 7 degrees C or 1.25 days of incubation at 10 degrees C, respectively . Cultures that were maintained in the log phase at 12 degrees C became increasingly heterogeneous in cell size after 2 days, but cultures that were maintained at 13 degrees C remained constant in cell size for 8 days . The observations have implications for the prediction of mesophile proliferation at temperatures that approach their minima for growth. Water Res, 2003 Sep, 37(16), 3921 - 7 Formulation of a mathematical model to predict solar water disinfection; Salih FM; A mathematical model was formulated that will facilitate the prediction of solar disinfection by analyzing the effect of sunlight exposure (x(1)) and the load of bacterial contamination (x(2)), as predictor variables, on the efficiency of solar disinfection (y) . Aliquots of 0.1 ml containing average numbers of E . coli, ranging between 1 and 5 x 10(3)cells/ml raw water, were introduced into each of the 96 wells of polystyrene microtitre plates . Plates, with the lid on, were exposed to sunlight for varying exposures ranging between 1.04 x 10(3) and 8.40 x 10(3)kJ m(-2) . Double strength nutrient broth was then added . After 48 h incubation wells containing visible contamination were considered as containing one cell or more that survived the exposure . Data showed that disinfection is dependent both on the load of bacterial contamination and sunlight exposure . This relationship is characterized by curves having shoulders followed by a steep decline and then tailing off in an asymptotic fashion . The shoulder size increased with the increase of the contamination load, however, the slope remains the same . Statistical analysis indicates a positive correlation among the variables (R(2) = 0.893); the mathematical model, y=1-(1-e(-kx(1)))(x(2)), represents the relationship, with k being the solar inactivation constant . The exposure required to produce a given decontamination level can be predicted using the equation: x(1)=-1/kln{1-(1-y)(-1/x(2))}e(-micro/rho.m/A), where micro is the linear attenuation coefficient (m(-1)), rho is the density, m is the mass and A is the area of the exposed part of the sample . The predictor variables (x(1), x(2)) strongly influence the efficiency of solar disinfection, which can be predicted using the suggested mathematical model . The present data provides a means to predict the efficiency of solar disinfection as an approach to improve the quality of drinking water mainly in developing countries with adequate sunshine all year-round. J Bacteriol, 2003 Aug, 185(15), 4450 - 60 CsrA regulates translation of the Escherichia coli carbon starvation gene, cstA, by blocking ribosome access to the cstA transcript; Dubey AK et al.; CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence . The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia coli genomic sequence for other genes that might be regulated by CsrA . cstA contained an exact match that overlapped its Shine-Dalgarno sequence . cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter . Expression of a cstA'-'lacZ translational fusion in wild-type and csrA mutant strains was examined . Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose . It was previously shown that cstA is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by Esigma(70) . We investigated the influence of sigma(S) on cstA expression and found that a sigma(S) deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained . The mechanism of CsrA-mediated cstA regulation was also examined in vitro . Cross-linking studies demonstrated that CsrA is a homodimer . Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts . RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA Shine-Dalgarno sequence, as predicted . These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding. Biotechnol Prog, 2003 Mar-Apr, 19(2), 659 - 61 Broth recycling to reduce process noise resulting from concentrated substrate addition in fed-batch cultivation of Escherichia coli; Johnston WA et al.; In this work feed hardware for fed-batch cultivation is presented (broth recycle feed injection system or BRFIS) . BRFIS proved superior to conventional submerged or dripped feed systems in reducing dissolved oxygen (DO) oscillations during Escherichia coli fed-batch cultivation (5 min coefficient of variation of 0.7% for BRFIS as compared to 26% or greater for conventional feeding hardware in a 2 L test reactor) . Hence, BRFIS is useful for fed-batch cultivation systems where the DO signal is used in measurement or control. Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 718 - 23 {High density fed-batch culture of Escherichia coli DH5 alpha/pDH-B2m with DO feed-back control of nutrient feeding}; Li Y et al.; Optimization of cultivation condition of recombinant E . coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2 . The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E . coli. Acta Biochim Pol, 2003, 50(1), 239 - 47 Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein; Ochocka AM et al.; In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis . We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1 . Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used . Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB . Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h . The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5 . Under these conditions over 90% of the fusion protein was present in a soluble form . The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns . The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E . coli culture. Mol Cells, 2003 Feb 28, 15(1), 20 - 6 The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity; Chang HS et al.; The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance . The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3) . It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth . Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa . When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min . In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously . Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha) . Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats. Infect Immun, 2003 Apr, 71(4), 2120 - 9 Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells; Zhou X et al.; The type III secretion system (TTSS) of enteropathogenic Escherichia coli (EPEC) has been associated with the ability of these bacteria to induce secretion of proinflammatory cytokines, including interleukin-8 (IL-8), in cultured epithelial cells . However, the identity of the effector molecule directly involved in this event is unknown . In this study, we determined that the native flagellar filament and its flagellin monomer are activators of IL-8 release in T84 epithelial cells . Supernatants of wild-type EPEC strain E2348/69 and its isogenic mutants deficient in TTSS (escN) and in production of intimin (eae), grown in Luria-Bertani broth, elicited similar amounts of IL-8 secretion by T84 cells . In contrast, supernatants of EPEC fliC mutants and of B171, a nonflagellated EPEC strain, were defective in inducing IL-8 release, a phenotype that was largely restored by complementation of the fliC gene in the mutant lacking flagella . Purified flagella from E . coli K-12, EPEC serotypes H6 and H34, and enterohemorrhagic E . coli serotype H7 all induced IL-8 release in T84 cells . Induction of IL-8 by purified flagella or His-tagged FliC from EPEC strain E2348/69 was dose dependent and was blocked by a polyclonal anti-H6 antibody . Finally, the mitogen-activated protein kinases (Erk1 and -2 and Jnk) were phosphorylated in flagellin-treated T84 cells, and inhibition of the p38 and Erk pathways significantly decreased the IL-8 response induced by EPEC flagellin . Our data clearly indicate that FliC of EPEC is sufficient to induce IL-8 release in T84 cells and that activation of the Erk and p38 pathways is required for IL-8 induction. Biotechnol Appl Biochem, 2003 Aug, 38(Pt 1), 9 - 13 Purification of soluble human epidermal growth factor (hEGF) from recombinant Escherichia coli culture broth by using expanded-bed adsorption chromatography; Lee YS et al.; Human epidermal growth factor (hEGF) secreted by recombinant Escherichia coli was purified from culture broth by expanded-bed adsorption (EBA) chromatography, strong anion-exchange chromatography and finally preparative reversed-phase HPLC (RP-HPLC) . The EBA chromatography step simultaneously captured the hEGF by cationic exchanger and removed the cellular biomass from the diluted culture broth . This step was carried out at high throughput, and resulted in a high yield (>90%) and a purification factor of approx . 20-fold to >80% purity . Its process performance was well maintained during a 16-fold scale-up . After the successive purification steps of anion-exchange chromatography and RP-HPLC, the overall yield was approx . 84% and the purity was satisfactory (>99.5%) . It was concluded that the purification process was very efficient and scaleable, warranting its implementation in large-scale manufacturing. J Infect Chemother, 2002 Dec, 8(4), 345 - 8 Enteroaggregative Escherichia coli: incidence in Japan and usefulness of the clump-formation test; Iwanaga M et al.; The usefulness of the clump-formation test described by Albert et al . for identifying enteroaggregative Escherichia coli (EAggEC) and the incidence of EAggEC in Japan were studied . One hundred and seventy strains of E . coli agglutinated with enteropathogenic E . coli diagnostic antisera were collected from a variety of districts in Japan . All isolates were from diarrheal stools . EAggEC was identified on the basis of the presence of the aggR gene accompanied by aggregative adhesion to HEp-2 cells . After 24 strains carrying eaeA, elt, est, stx-1, stx-2, or ipaH genes were eliminated, the remaining 145 strains were examined for adhesion to Hep-2 cells, the presence of the aggRgene, and clump formation on the surface of Muller-Hinton broth . aggR was detected in 10 strains, and 9 of them displayed aggregative adhesion to HEp-2 cells . Seven strains produced marked clumps and 22 showed moderate clump formation . The sensitivity and specificity of the clump-formation test for detecting EAggEC were each about 90%, and they varied slightly depending on the stringency of evaluation for the degree of clump formation . From these results, we conclude that the incidence of EAggEC cannot be ignored as a possible cause of diarrheal disease in Japan, and we strongly recommend the clump-formation test for detecting EAggEC. J Food Prot, 2002 Dec, 65(12), 1943 - 8 Utilization of fluorogenic assay for rapid detection of Escherichia coli in acidic fruit juice; Pao S et al.; This study was undertaken to investigate interference by acids commonly found in fruit juice in Escherichia coli assays involving the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) as a fluorogenic substrate for enzyme reaction . Fluorescence intensity was negatively correlated (P < 0.001) with the volume of fresh citrus juice tested by the lauryl tryptose broth (LST)-MUG assay, and the permissible sample sizes were limited to 0.3 and 0.5 ml for fresh citrus juices with pHs of 3.3 and 3.9, respectively . In addition, false-negative results were visually observed under UV light when the E*Colite assay was used to test large volumes (5 to 10 ml per test) of fresh citrus juice or when the test broth used for the LST-MUG assay was supplemented with citric, malic, or tartaric acid at 2 to 4 g/liter . These results suggest that the size and pH of acidic samples should be controlled in MUG-based fluorogenic assays . The inhibitory effect on fluorescence was due to high acidity, which reduces fluorescence from 4-methylumbelliferone . Buffering improved the assays . When sodium bicarbonate was incorporated in the enrichment broth at 10 g/liter, the permissible sample sizes for fresh grapefruit juice (pH 3.1) increased from 0.3 to 1 ml for the LST-MUG (with 9.9 ml of broth) assay and from 3 to 10 ml for the E*Colite (with 99 ml of broth) assay. J Protein Chem, 2002 Aug, 21(6), 413 - 8 Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli; Zhao F et al.; Expression of fusion protein trypsin-streptavidin (TRYPSA) in Escherichia coli was evaluated and the protein purified . Protein expression was induced by 1 mM isopropylthio-beta-D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-L-arginine methyl ester (TAME) . Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased . The total expression in Luria-Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30 degrees C . The optimum expression level was 35 and 48 U/L in LB and TB, respectively . Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature . The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC . A molecular weight of 39-40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation. Appl Microbiol Biotechnol, 2002 Dec, 60(4), 408 - 16 Epub 2002 Nov 05. Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3); Ramchuran SO et al.; Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli . This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-beta- D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations . Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E . coli strain BL21 (DE3) . A three-fold difference in the specific activity of both xylanase variants {between 7,000 and 21,000 U/(g cell dry weight)}, was observed under the different conditions tested . Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post-induction phase and simultaneously protease activity was detected . Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l(-1)), as well as in induced cultivations employing metabolite-supplemented nutrient feeds . By contrast, maximum specific cellulase activity {between 700 and 900 U/(g cell dry weight)} remained relatively unaffected in all cases . The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed. Int J Food Microbiol, 2003 Mar 15, 81(2), 113 - 21 Enumeration of coliforms and Escherichia coli in frozen black tiger shrimp Penaeus monodon by conventional and rapid methods; Suwansonthichai S et al.; Conventional (most probable number, MPN) and rapid methods-including Chromocult coliform agar (CCA), Fluorocult(R) LMX broth (LMX), and Petrifilm Escherichia coli count plates (PEC) for enumeration of coliforms and E . coli in frozen black tiger shrimp from Thailand were compared in order to assess the possibility of using one of the rapid methods for routine analysis . Enumeration of coliforms and E . coli from 18 samples of regular frozen black tiger shrimp and 156 samples of frozen black tiger shrimp experimentally contaminated with coliforms or E . coli at concentrations of approximately 10, approximately 10(2), and approximately 10(3) CFU g(-1) revealed that at the level of approximately 10 CFU g(-1), coliform numbers ranked as LMX>CCA>MPN=PEC and E . coli as MPN=LMX=PEC=CCA . At the level of approximately 10(2) CFU g(-1), coliform numbers ranked as LMX>MPN=PEC=CCA and E . coli as MPN=LMX>PEC=CCA . At the level of 10(3) CFU g(-1), coliforms ranked as LMX>MPN=CCA>PEC and E . coli as MPN>LMX>CCA>PEC . Agreements with the conventional MPN method for coliforms were LMX 108%, PEC 87.2%, and CCA 91.2% and agreements for E . coli were LMX 101%, PEC 95.7%, and CCA 96.3% . Sensitivities (%) ranked LMX>MPN>CCA=PEC for coliforms and E . coli, whereas equal specificities (100%) of all methods for coliforms and E . coli were demonstrated . Rankings for the other parameters compared were: convenience, PEC>CCA=LMX>MPN; time to detection, MPN>LMX=PEC=CCA; expense, MPN=PEC>CCA>LMX; labor, MPN>LMX=CCA>PEC; accuracy for coliforms, PEC>CCA>MPN>LMX; and accuracy for E . coli, PEC=CCA>LMX>MPN. J Appl Microbiol, 1997 Mar, 82(3), 301 - 9 Relationship between respiratory enzymes and survival of Escherichia coli under starvation stress in lake water; Ozkanca R et al.; Survival, electron transport system (ETS) activity and the activity of NADH and succinate dehydrogenase of Escherichia coli ML30 were studied under starvation stress at different temperatures in a filtered-autoclaved lake water microcosm . ETS activity in E . coli declined rapidly at 30 degrees C but more slowly at 4 degrees and 15 degrees C over a 20 d starvation period . The decrease in ETS activity in E . coli only started after 6 d of incubation at 4 degrees C and 15 degrees C . Viability of E . coli, as determined by plate counts, declined faster at 37 degrees C than at the other temperatures and remained highest at 4 degrees C in filtered-autoclaved lake water . There was also a significant cell size reduction at 37 degrees C in filtered-autoclaved lake water but not at 4 degrees C . ETS activity after up to 16 d of starvation increased after the addition of nutrient broth to the filtered-autoclaved lake water at 15 degrees C and 30 degrees C suggesting that cells were still able to respond to nutrients, even after prolonged starvation . The response to the addition of nutrient broth, however, declined with the length of the starvation period . The activity of both succinate and NADH dehydrogenase declined over a 13 d starvation period . The loss of activity was fastest at 37 degrees C compared to lower incubation temperatures but even at 4 degrees C, a significant proportion of the activity was lost over the 13 d period. Infect Immun, 2002 Nov, 70(11), 6094 - 106 Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophila and its role in detoxification of lysophosphatidylcholine; Flieger A et al.; We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids . In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids . The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL . In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol . An L . pneumophila plaA mutant was generated by allelic exchange . Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L . pneumophila . The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA . Overexpression of plaA completely protected L . pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids . The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L . pneumophila . In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L . pneumophila. Mol Microbiol, 2002 Oct, 46(1), 281 - 91 Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12; Oshima T et al.; We have systematically examined the mRNA profiles of 36 two-component deletion mutants, which include all two-component regulatory systems of Escherichia coli, under a single growth condition . DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria-Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes . Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes . Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two-component systems . A high correlation coefficient of expression profile was found between several two-component mutants . Together, these results support the view that a network of functional interactions, such as cross-regulation, exists between different two-component systems . The compiled data are avail-able at our website . Infect Immun, 2002 Oct, 70(10), 5659 - 69 Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection; Robey M et al.; In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b . As it is in Escherichia coli, the L . pneumophila feoB gene was contained within a putative feoAB operon . L . pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type . Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L . pneumophila FeoB . However, the L . pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans . In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2'-dipyridyl . The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth . Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages . The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB . The L . pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice . Taken together, these results indicate that L . pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments . These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen. J Bacteriol, 2002 Aug, 184(15), 4246 - 58 pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli; Stancik LM et al.; Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors . Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions . Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1 . 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent . At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE) . These responses could be mediated by the reuptake of acetate driven by changes in pH . The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC . In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB . pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7 . Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced) . The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator . The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility . High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT . These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines . Overall, these data are consistent with a model in which E . coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme. Lett Appl Microbiol, 2002, 35(2), 153 - 6 Nuclease fluorescence assay for the detection of verotoxin genes in raw milk; Burk C et al.; AIMS: To develop a rapid, high throughput PCR method for the detection of verotoxigenic Escherichia coli (VTEC) in raw milk based on TaqMan PCR . METHODS AND RESULTS: Two TaqMan PCR systems for the detection of verotoxin genes 1 and 2, respectively, have been established . A total of 74 bacterial strains, among them 15 VTEC, were used to characterize the PCR tests . No false negative and no false positive reactions were observed . When artificially contaminated raw milk samples of 25 ml were cultured in enrichment broth for 24 h, inocula of 10(-1) cells ml-1 could be detected . CONCLUSIONS: The TaqMan PCR systems are feasible for the detection of VTEC in raw milk . SIGNIFICANCE AND IMPACT OF THE STUDY: The TaqMan PCR offers a rapid semiautomated alternative to conventional PCR methods for the detection of VTEC in raw milk. Appl Environ Microbiol, 2002 Jul, 68(7), 3377 - 84 Technical-scale production of cyanophycin with recombinant strains of Escherichia coli; Frey KM et al.; By the use of Escherichia coli DH1 harboring cphA from Synechocystis sp . strain PCC6803, large-scale production of cyanophycin at 30- and 500-liter culture volumes was established . Transcription of cphA was controlled by the thermosensitive cI857 repressor, which enabled induction of cphA by a simple temperature shift in the culture fluid . Maximum cyanophycin cell content of up to 24% (wt/wt) of cellular dry matter was obtained by induction in the early exponential growth phase and cultivation of the cells in terrific broth complex medium . Synthesis of cyanophycin was found to be strongly dependent on the presence of complex components, and in mineral salts medium the cells synthesized and accumulated cyanophycin only if Casamino Acids were added . Cultivations were done at the 500-liter scale, allowing the provision of cell mass for the preparation of cyanophycin at the kilogram scale . Isolation of cyanophycin was achieved by a new acid extraction procedure which allowed large-scale purification of the polyamide from whole cells. J Biol Chem, 2002 Jul 5, 277(27), 24155 - 61 Epub 2002 Apr 24. EnvZ-OmpR interaction and osmoregulation in Escherichia coli; Cai SJ et al.; EnvZ, a histidine kinase/phosphatase in Escherichia coli, responds to the osmolarity changes in the medium by regulating the phosphorylation state of the transcription factor OmpR, which controls the expression levels of outer membrane porin proteins OmpF and OmpC . Although both ompR and envZ genes are located on the ompB locus under the control of the ompB promoter and transcribed as a single polycistronic mRNA, the expression of envZ is known to be significantly less than ompR . However, to date no accurate estimation for the amounts of EnvZ and OmpR in the cell has been carried out . Here we examined the levels of EnvZ and OmpR in the wild-type strain MC4100 by quantitative Western blot analysis using anti-OmpR and anti-EnvZc (cytoplasmic domain of EnvZ) antisera . It was observed that during exponential growth in L-broth medium there were approximately 3500 and 100 molecules per cell of OmpR and EnvZ, respectively . The levels of OmpR and EnvZ in MC4100 cells grown in a high osmolarity medium (nutrient broth with 20% sucrose) were about the same as those grown in L-broth, whereas they were 1.7-fold higher than those in a low osmolarity medium (nutrient broth) . With His10-OmpR, we also determined that the K(d) value for the EnvZc-OmpR complex formation is 1.20 +/- 0.17 microm . On the basis of these results, the molecular mechanism of osmoregulation of ompF and ompC is discussed. Lett Appl Microbiol, 2002, 34(4), 274 - 8 Comparison of LST + MUG broth technique and conventional method for the enumeration of Escherichia coli in foods; Dogan HB et al.; AIMS: To reduce the analysis time needed for the enumeration of Escherichia coli, a rapid fluorogenic method (MUG) which takes only 48 h was compared with the standard most probable number (MPN) method which takes 6 days as described in the International Standards Organization (ISO) . This study provides reliability data for the fluorogenic method applied to certain foods . METHODS AND RESULTS: Both methods were applied to 500 food samples which were analysed for E . coli enumeration . Agreement between the two methods was found in 409 (81 x 8%) samples; 81 (16 x 2%) samples gave higher values by the fluorogenic method, and only 10 (2 x 0%) samples were more effectively assayed by the ISO method . According to statistical analysis, the reliability between the methods was r = 0 x 9706, r(2) = 0 x 9421 and Cronbach's alpha = 0 x 9851 . While all three values showed a high degree of correlation (P < 0 x 0001) between the two methods, McNemar's test demonstrated a significant difference between them, indicating that the MUG method was more reliable than the ISO method . CONCLUSIONS: The data suggest that the fluorogenic method is more reliable and shorter to perform than the standard ISO method . SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of the two methods may provide a rapid and more reliable alternative for the enumeration of E . coli in food samples. Lett Appl Microbiol, 2002, 34(4), 269 - 73 The correlation method for rapid monitoring of Escherichia coli in foods; Gray PM et al.; AIMS: A new rapid method was developed to rapidly monitor Escherichia coli counts in foods . MATERIALS AND RESULTS: One ml of modified selective broth with 4-methylumbelliferyl beta-D-glucuronide and 1 ml of food sample were mixed in a sterile test tube and incubated at 37 degrees C . The positive reaction (fluorescence under u.v . light) was monitored at regular 30 min intervals . The positive reaction times in test tubes were compared with actual E . coli numbers from tested samples . The growth of E . coli in test tubes (broth) was much faster than growth on agar . The first experiment was performed to evaluate the rapid correlation method using pure E . coli cultures . The correlation between E . coli counts by the conventional plating method and positive reaction (fluorescence production) times in test tubes was highly agreeable (r(2) = 0 x 95) . In the case of low E . coli numbers, such as 2 x 0 log10 cfu ml(-1), the rapid correlation method detected their presence after 10 h incubation . When highly contaminated samples were assayed (8 log10 cfu ml(-1)), the rapid correlation method detected the presence of E . coli after 4 h incubation . In the ground beef experiment, the correlation between fluorescence production time and actual E . coli numbers was also strongly agreeable (r(2) = 0 x 92) . CONCLUSIONS: From these results, it is obvious that the new rapid method can rapidly monitor E . coli counts in foods . SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the new method saved about 10-14 h incubation time compared to conventional plating methods . The rapid correlation method required much shorter incubation times compared to conventional plating methods for monitoring E . coli. J Bacteriol, 2002 Apr, 184(8), 2192 - 203 Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator; Haydel SE et al.; The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators . Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M . tuberculosis . Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M . tuberculosis growth in cultured human primary macrophages . To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli . Upon induction of the TrcR protein, there was a >500-fold increase in beta-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation . Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding . By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding . Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays . To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR . Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR . This report indicates that the M . tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter. J Biotechnol, 2002 Mar 28, 94(2), 185 - 94 Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli; Vallejo LF et al.; Eschericha coli was genetically engineered to produce recombinant human bone morphogenetic protein-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system . High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 gl(-1)) were obtained by applying a high-cell-density cultivation procedure . After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to 100 mgl(-1) by means of a simple dilution method with yields exceeding 50% . Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer . With the established renaturation and purification protocols, yields of more than 10 mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth . The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells. Anal Chem, 2001 Dec 15, 73(24), 5866 - 74 Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time; Ozawa T et al.; In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria . We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al . Anal . Chem . 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP . Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore . This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells . In this paper, we have explored the use of the DnaE split intein from Synechocystis sp . PCC6803 for intracellular reconstitution of the split EGFP . We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions . A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein . The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates . This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest. Int J Food Microbiol, 2001 Dec 4, 71(1), 101 - 4 Biosynthetic requirements for the repair of membrane damage in pressure-treated Escherichia coli; Chilton P et al.; Cells of Escherichia coli that survived pressure treatment at 400 MPa showed increased sensitivity to sodium deoxycholate or sodium chloride in the plating medium, implying that homeostatic or barrier functions associated with outer and cytoplasmic membranes, respectively, were impaired . Repair of such sublethal membrane damage occurred when cells were incubated at 37 degrees C in tryptone soya broth . Inhibitor studies indicated that repair of cytoplasmic membrane damage was energy-dependent and required RNA and protein synthesis, whereas repair of outer membrane damage occurred with no requirement for energy or RNA or protein synthesis. Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 131 - 7 Establishment of a hyper-protein production system in submerged Aspergillus oryzae culture under tyrosinase-encoding gene (melO) promoter control; Ishida H et al.; UV-mediated mutagenesis generated a high glucoamylase-producing mutant of Aspergillus oryzae exhibiting strong melanization in solid-state culture . Expression of the glucoamylase-encoding gene (glaB), which is specifically expressed in solid-state culture, and the tyrosinase-encoding gene (melO), was analyzed using an E . coli beta-glucuronidase (GUS) reporter assay to investigate this phenomenon . Although no common regulation was found for melO and glaB expression, the former was greatly enhanced in submerged culture . Interestingly, the melO promoter was about four times stronger for GUS production than the powerful promoters amyB, glaA, and modified agdA, previously isolated for industrial heterologous gene expression in A . oryzae . These findings indicated that the melO promoter would be suitable for hyper-production of heterologous protein in Aspergillus . The glaB-type glucoamylase selected as the target protein was produced in a submerged culture of A . oryzae under the control of the melO promoter . The maximum yield was 0.8 g/l broth, and the total extracellular protein purity was 99% . Repeated batch culture, to improve productivity, gave a maximum yield of 3.3 g/l broth . The importance of this work is in the establishment of a both high-level and high-purity protein overproduction system in A . oryzae by use of the melO promoter. Biotechnol Bioeng, 2001 Nov 20, 75(4), 451 - 5 Direct chemical extraction of a recombinant viral coat protein from Escherichia coli at high cell density; Choe WS et al.; The release of protein and DNA from nonrecombinant E . coli JM101 and recombinant E . coli HMS174(DE3) expressing L1 (the major viral coat protein of human papillomavirus type 16) as an inclusion body was demonstrated at high cell density (OD(600) = 160) . For the nonrecombinant strain, extraction efficiency decreased significantly as cell mass increased, with a high viscosity increase in the postextraction broth . A different dependence on cell concentration was observed for the recombinant strain, with total protein extraction efficiency exceeding 85% for both uninduced and induced cells . Almost complete release of the recombinant L1 protein was achieved at high cell concentration (OD(600) = 80 approximately 160) without the use of reducing agent . This greatly extends the concentration range for chemical extraction . Carbohydr Res, 2001 Sep 21, 335(1), 11 - 21 Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase; Sawabe T et al.; A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia coli . Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues . The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29 . A region G(165) to V(194) in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P . elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (pI 4.78) of the region resembled those of the purified enzyme . Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E . coli in LB broth containing 50% (v/v) artificial seawater (ASW) . Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products . Subcloning of the region G(165) to N(398) of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme . A region A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain of Streptomyces chitinases, but did not exhibit any catalytic activity. J Am Water Works Assoc, 1997 Sep, 89(9), 112 - 20 Comparative performance of Colisure; McFeters GA et al.; Colisure presence-absence medium was compared with standard reference methods for detecting low numbers of total coliform bacteria and E . coli in drinking water when the bacteria were subjected to chlorine stress . When Colisure was compared with established reference methods to detect total coliforms in dilute, disinfected samples, Colisure yielded more positive results after 24, 28, and 48 h than lauryl tryptose broth (LTB) confirmed in bile green lactose broth after 48 h . Colisure also detected higher levels of chlorine-injured E . coli than LTB confirmed in EC medium with 4-methylumbelliferyl B-D-glucuronide (EC/MUG) . The sensitivity and specificity of Colisure were also evaluated and were determined to be between 96 and 100 percent on nonchlorinated samples when positive and negative tests were verified. Mol Biotechnol, 2001 Jul, 18(3), 269 - 73 High level of expression of the Toxoplasma gondii-recombinant Rop2 protein in Escherichia coli as a soluble form for optimal use in diagnosis; Nigro M et al.; The rhoptry 2 protein (Rop2) is an interesting protein of Toxoplasma gondii that is involved in the parasite invasion of host cell, it has three T-cell epitopes and high antigenic value . However, the expression of Rop2 as a recombinant protein in Escherichia coli is not an easy task, showing low levels of expression or degradation and solubility problems . Using a recombinant Rop2(196-561) fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth . rRop2(196-561) was purified mainly as a soluble product and in high concentrations (approx 1 mg/mL) under native conditions (40 mM imidazol in phosphate buffer) . However, after a cycle of freezing-thawing rRop2(196-561) became insoluble . When glycerol was added to 26%, immediately after purification, the protein stayed soluble after cycles of freezing-thawing . Finally, it was demonstrated that under these conditions soluble rRop2(196-561) keeps its diagnostic value in contrast with the insoluble protein. Curr Microbiol, 2001 Sep, 43(3), 215 - 9 Differences in Escherichia coli culture conditions can have a large impact on the induction of extreme acid resistance; Jarvis GN et al.; A recently isolated Escherichia coli strain (3TF4) survived an acid shock that mimicked the low pH of the human gastric stomach (pH 2, 1 h), but this survival was highly influenced by prior growth conditions . Only 0.01% of the stationary phase cells that had been grown anaerobically in a carbonate medium (2 mg glucose and 0.25 mg yeast extract per ml, 40 mm sodium carbonate, final pH 6.5) survived the acid shock, and the survival of exponential phase cultures was even lower (0.0001%) . Small amounts of Trypticase (1.5 mg/ml) increased the survival as much as 5000-fold, but cultures that were provided with higher concentrations of Trypticase (7.5 mg/ml) did not reach the stationary phase in 24 h and were more acid sensitive . Sodium acetate (50 mm) also increased acid resistance, and the increased acid shock survival was greater for the cells that had reached the stationary phase (100 versus 1000-fold, respectively) . E . coli 3TF4 cultures that had been grown aerobically in Luria broth were already so acid resistant (survivals greater than 40%) that they did not respond to sodium acetate . E . coli 3TF4 cultures that were refrigerated (5 degrees C, 7 days) were nearly as acid resistant as those that were immediately subjected to acid shock (pH 2.0, 1 h). Lett Appl Microbiol, 2001 May, 32(5), 303 - 6 Comparative study of the influence of melatonin and vitamin E on the surface characteristics of Escherichia coli; Uberos J et al.; AIMS: Melatonin is a hormone produced by the pineal gland and that affects the response of various cell membranes to an oxidative stimulus . METHODS AND RESULTS: The present study evaluates the hydrophobic characteristics of Escherichia coli in response to melatonin (100 nmol l(-1), 200 micromol l(-1)) and to vitamin E (5 mg dl(-1)) . A reduction was found in the surface hydrophobicity of E . coli at concentrations of 200 micromol l(-1) melatonin in a Mueller-Hinton (MH) broth . These effects were modified when a protein synthesis inhibitor (chloramphenicol) was added at sub-lethal concentrations to the broth . Vitamin E produced a greater diminution in surface hydrophobicity than melatonin . The adherence of E . coli to nitrocellulose filters increased in the presence of melatonin + chloramphenicol, and vitamin E . The effects observed were independent of the concentration of iron in the broth . CONCLUSION: Oxidative stress plays an important role in modifying the surface characteristics of E . coli, which could affect the micro-organism's capacity to adhere to epithelia . SIGNIFICANCE AND IMPACT OF THE STUDY: We think that the oxide reduction potential of the host may be a determinant factor in the bacterial colonization of animal tissue. Res Microbiol, 2001 Jan-Feb, 152(1), 17 - 26 Survival of Escherichia coli during long-term starvation: effects of aeration, NaCl, and the rpoS and osmC gene products; Conter A et al.; The survival of Escherichia coli was investigated during long-term starvation in rich media . In aerated cultures, E . coli lost the ability to form colonies earlier in NaCl-free Luria broth than in LB medium containing NaCl . Improved survival at low aeration and the sensitivity to hydrogen peroxide in aging cultures indicated a major role for oxidative stress in cell mortality . Mutants in rpoS, lacking the sigmaS subunit of RNA polymerase, showed altered survival in salt-containing media . However, in the absence of NaCl, although these mutants exhibited a massive loss of viability during the first 2 days, this was followed by a stabilization of the number of survivors . The starved culture contained survivors until at least day 9, long after a wild-type strain had completely lost viability . This peculiar behavior suggests that, in rich media of low osmotic pressure, sigmaS helps in short-term survival but hampers long-term survival . Mutants in osmC, a member of the rpoS regulon, also exhibited reduced survival and increased sensitivity to oxidative stress . The biochemical function of the envelope protein OsmC remains unknown, but present data indicated that it participates, directly or indirectly, in the defense against oxidative compounds. J Bacteriol, 2001 Feb, 183(4), 1242 - 7 Novel genes affecting urease acivity in Actinobacillus pleuropneumoniae; Bosse JT et al.; Characterization of a series of urease-negative transposon mutations of Actinobacillus pleuropneumoniae revealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster . A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes . As well, a partial ORF, apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation . The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators . Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system . The cbiM and cbiQ genes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and the cbiO gene encodes a cobalt transport ATP-binding protein homologue . The product of the cbiK gene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein . Escherichia coli clones containing this putative transport operon together with the urease genes of A . pleuropneumoniae were urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl(2) for full urease activity . This result supports the hypothesis that nickel is a substrate for this permease system . The sixth ORF, utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function in A . pleuropneumoniae remains to be determined. J Antibiot (Tokyo), 2000 Sep, 53(9), 954 - 8 Cyclo(dehydroala-L-Leu), an alpha-glucosidase inhibitor from Penicillium sp . F70614; Kwon OS et al.; A diketopiperazine (1) has been isolated from the culture broth of Penicillium sp . F70614 and its structure has been determined to be cyclo(dehydroala-L-Leu) by various spectroscopic analyses . This compound selectively inhibited yeast alpha-glucosidase and porcine intestinal alpha-glucosidase with IC50 values of 35 and 50 microg/ml, respectively . However, it did not show significant inhibitory effects against almond beta3-glucosidase, Aspergillus alpha-galactosidase, Escherichia coli beta-galactosidase and jack bean alpha-mannosidase. Int J Food Microbiol, 2000 Nov 1, 61(2-3), 159 - 67 Modelling the combined temperature and salt (NaCl) limits for growth of a pathogenic Escherichia coli strain using nonlinear logistic regression; Salter MA et al.; A broth-based method is used to determine if exponential phase Escherichia coli R31, an STEC, is able to grow within 50 days under various combinations of sub-optimal temperatures and salt concentrations . From these data, the growth limits for combinations of temperature (7.7-37.0 degrees C) and water activity (0.943-0.987; NaCl as humectant) are defined and modelled using a nonlinear logistic regression model . That form of model is able to predict the combinations of salt concentration/water activity and temperature that will prevent the growth of E . coli R31 with selected levels of confidence . The model fitted the data with an approximate concordance rate of 97.3% . The minimum water activity that permitted growth occurred in the range 25-30 degrees C, the temperature range which optimises cell yield . At temperatures below this range the minimum water activity which allowed growth increased with decreasing temperature. Avian Dis, 2000 Jul-Sep, 44(3), 545 - 8 Importance of Escherichia coli infection in ascites in broiler chickens shown by experimental production; Yamaguchi R et al.; Common commercial strain male broilers aged 14 days were intratracheally inoculated with 0.2 ml of 1.2 x 10(6) colony-forming units of Escherichia coli in nutrient broth and kept in a cool environment during the experiment . Ascites was produced in five surviving and two dead birds out of 50 but not in 50 mock-infected control birds . Among the 40 survivors that were infected, the erythrocyte packed cell volume (PCV) of the 10 birds with pericarditis was the same as in 21 grossly normal birds, although that of the four birds with enlarged right ventricle (RV) was high . The pericarditis caused by E . coli septicemia was not the primary cause of ascites . However, the PCV was high in some of the survivors with an enlarged RV without pericarditis, indicating overload due to the lung lesion . These data suggested that some of the birds with an enlarged RV, caused by supplying blood that was insufficiently oxygenated for the body size, suffered from ascites. Appl Microbiol Biotechnol, 2000 Jun, 53(6), 655 - 60 Production of interferon-alpha in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins; Babu KR et al.; Escherichia coli TG1 transformed with a temperature-regulated interferon-alpha expression vector was grown to high cell density in defined medium containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process . Feeding was carried out to achieve an exponential increase in biomass at growth rates which minimized acetate production . Thermal induction of such high cell density cultures resulted in the production of approximately 4 g interferon-alpha/l culture broth . Interferon-alpha was produced exclusively in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose . The yield of purified interferon-alpha was approximately 300 mg/l with respect to the original high cell density culture broth (overall yield of approximately 7.5% active interferon-alpha) . The purified recombinant interferon-alpha was found by different criteria to be predominantly monomeric and possessed a specific bioactivity of approximately 2.5 x 10(8) IU/mg based on viral cytopathic assay. J Food Prot, 2000 Apr, 63(4), 539 - 41 Efficacy of chromocult coliform agar for coliform and Escherichia coil detection in foods; Turner KM et al.; Chromocult coliform agar (CCA) was compared with Petrifilm Escherichia coli count plate (PEC) for identifying coliforms and E . coli in a variety of meat products . Products examined included 45 raw beef samples, 12 sausage emulsion samples, 11 samples of meat-based ready-to-eat appetizers, and 8 pork trimming samples . Coliforms from CCA and PEC were confirmed by gassing in brilliant green lactose broth plus a positive reaction on purple broth agar plus lactose after incubation at 35 degrees C for 48 h . Lauryl sulfate tryptose plus methylumbelliferyl-beta-glucuronide and tryptophan broth were used to confirm E . coli from CCA and PEC with 48-h incubations at 35 and 42.5 degrees C, respectively . API 20E test strips were inoculated for final confirmation . The overall respective confirmation percentages (CFU/g) for the PEC and the CCA methods were 93.1 and 93.7% for coliforms and 99.8 and 98.1% for E . coli, although the CCA method yielded significantly (P < 0.001) higher mean CFU/g values for both coliforms and E . coli . Regression analyses of these data indicated a strong positive linear relationship existed between the two methods over a wide CFU/g range for both coliforms and E . coli . The respective correlation coefficients obtained for coliforms and E . coli of 0.89 and 0.86 indicate that the CCA method provides a reliable optional method for these determinations in meat products. J Food Prot, 2000 Apr, 63(4), 534 - 8 Rapid detection of enterotoxigenic Escherichia coli O6 in water by using monoclonal antibody and a photon-counting television camera; Trevanich S et al.; Monoclonal antibodies (MAbs) raised against Escherichia coli O6:H16 were screened against 15 strains of E . coli and 19 non-E . coli bacteria . A MAb-luminescence assay using MAb-5.8, which shows no cross-reactions with non-E . coli bacteria, and a photon-counting television camera were developed for rapid enumeration of E . coli O6:H16 in water . The membrane filter that retained bacteria was boiled for 5 min in a buffer and incubated with biotinylated MAb-5.8 . After incubation with streptavidin-peroxidase conjugate, it was reacted with luminol-based reaction mixture . Luminous image and light intensity of the filter was recorded with a Biocell Counter . Levels of E . coli O6 higher than 7 x 10(3) CFU were detected by the MAb-luminescence assay when E . coli O6 was spotted onto the membrane filter . The sample that contained E . coli O6:H16 was filtered through a membrane filter, and the filter that retained bacteria was incubated on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl at 37 degrees C for 6 h . The number of light emission points on the filter correlated well with initial E . coli O6:H16 counts within the range of 1 to 3 x 10(2) CFU . The correlation coefficient was 0.89. Hum Antibodies, 1999, 9(3), 171 - 6 Studies on p53 immunolocalisation in breast cancer and its prognostic significance; Meenakshi A et al.; Immunocytochemical localisation of mutant p53 in breast tumours serves as a potential prognostic molecular marker . In order to study the expression of p53 protein in breast cancer which constitutes the second most common malignancy in the South Indian female population, MAb CIBCVMC12 has been generated against human p53 protein isolated and purified from bacterial cell lysate of E.coli carrying the plasmid T 7-7 Hup53 grown in Luria broth to induce the expression of p53 . The positive clones selected by ELISA were found to exhibit strong staining of nuclear p53 in both fresh and archival paraffin embedded breast tumour tissue sections . Commercial MAb D 07 against p53 was used as control . In immunoprecipitation, this MAb of IgG2b isotype was found to bind specifically to a protein of 53 kD . Immuno cyto chemical assay of normal, benign and malignant breast tissues of different histological types revealed that the majority of tumour cells were strong positive in the case of infiltrating ductal and lobular carcinomas, the staining being less intense for in situ carcinoma . The test for normal and benign tissues was negative . The staining patterns were comparable with those of control antibody . These results suggest that the MAb generated is specific to p53 . The p53 protein expression was compared with the estrogen receptor (ER) status for 50 breast tumours which revealed that 38% of these were p53 positive and of these two were ER+ . Among the p53 negative tumours, 48% were found to be ER+ . A comparison of the p53 expression for 100 breast cancer patients indicated that 57% of the tumours were p53 negative and these patients had a longer overall 5 year survival rate and recurrent free interval which is statistically very significant . These results might suggest that p53 positive tumours are more aggressive biologically with poor prognosis. J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 25 - 38 Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of Escherichia coli; Garke G et al.; Aggregation and precipitation are major pitfalls during bioprocessing and purification of recombinant human basic fibroblast growth factor (rh-bFGF) . In order to gain high yields of the soluble protein monomer with high biological activity, an efficient downstream process was developed, focussing on the combination of expanded bed adsorption (EBA) and heparin chromatography . After expression in E . coli TG1:plambdaFGFB, cells were harvested and washed; then the rh-bFGF was released via high pressure homogenization . The high viscosity of the feedstock of about 40 mPa s, showing non-newtonian behaviour, was reduced to 2 mPa s by the addition of DNase . The homogenate (5.6 l) was loaded directly on an expanded bed column (C-50) packed with the strong cation-exchanger Streamline SP . In the eluates, histone-like (HU) protein was identified as the main protein contaminant by sequence analysis . The thermodynamics and kinetics of rh-bFGF adsorption from the whole broth protein mixture were determined in view of competition and displacement effects with host-derived proteins . Optimal binding and elution conditions were developed with knowledge of the dependence of rh-bFGF adsorption isotherms on the salt concentration to allow direct application of eluates onto Heparin HyperD . This affinity support maintained selectivity and efficiency under CIP and over a wide range of flow-rates; both is advantageous for the flexibility of the purification protocol in view of a scalable process . Remaining DNA and HU protein were separated by Heparin HyperD . The endotoxin level decreased from approximately 1,000,000 EU/ml in the whole broth to 10 EU in 3 mg bFGF per ml . The final purification protocol yields >99% pure rh-bFGF as judged from SDS-PAGE and MALDI-TOF mass spectrometry with high mitogenic activity (ED50=1-1.5 ng/ml) of the lyophilized sample . In comparison to the conventional process, the overall protein recovery rose by 15% to 65% with saving time and costs. J Biol Chem, 2000 Feb 18, 275(7), 5081 - 9 The thioredoxin system of Helicobacter pylori; Windle HJ et al.; This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori . Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC . The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli . Trx was overexpressed by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein . The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds . H . pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/IgA/sIgA), and soluble mucin . Subcellular fractionation analysis of H . pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells . H . pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2',5'-ADP-agarose . Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis . H . pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5'-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H . pylori Trx . H . pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses . These observations suggest that Trx may conceivably assist H . pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction. Lett Appl Microbiol, 1999 Dec, 29(6), 375 - 9 Effect of post-treatment holding conditions on detection of tufA mRNA in ethanol-treated Escherichia coli: implications for RT-PCR-based indirect viability tests; Sheridan GE et al.; The PCR is a rapid and sensitive method for detecting and identifying low numbers of bacteria, but it does not discriminate between living and dead cells . Most messenger RNA (mRNA) molecules have a short half-life in the bacterial cell and their presence may therefore indicate viability . We have compared PCR and RT-PCR (targeted at tufA DNA or mRNA, respectively) for the detection of Escherichia coli, using healthy cells and those killed by exposure to different stress treatments . PCR gave a positive signal in live cells and those killed by autoclaving, boiling, or treatment with 50% ethanol, but was negative after exposure to pH 2.0 for 5 min . RT-PCR was positive in live cells but negative after all treatments except exposure to ethanol . The persistence of tufA mRNA was examined in ethanol-killed cells incubated in LB broth at different temperatures . The RT-PCR signal persisted for up to 16 h at 15 degrees C or 4 degrees C but disappeared within 2 h at 37 degrees C . RT-PCR thus has potential as an indicator of viability provided samples are pre-incubated under appropriate conditions that will ensure decay of any residual mRNA in dead cells. J Bacteriol, 2000 Jan, 182(2), 551 - 4 sigma(70) is the principal sigma factor responsible for transcription of acs, which encodes acetyl coenzyme A synthetase in Escherichia coli; Kumari S et al.; Cells of Escherichia coli undergo a metabolic switch associated with the production and utilization of acetate . During exponential growth on tryptone broth, these cells excrete acetate via the phosphotransacetylase-acetate kinase (Pta-AckA) pathway . As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively . This metabolic switch depends upon the induction of Acs . As part of our effort to dissect the mechanism(s) underlying induction and to identify the signal(s) that triggers that induction, we sought the sigma factor most responsible for acs expression . Using isogenic strains that carry a temperature sensitivity allele of the gene that encodes sigma(70) and either a wild-type or null allele of the gene that encodes sigma(S), we determined by immunoblotting, reverse transcriptase PCR, and acs::lacZ transcriptional fusion analyses that sigma(70) is the sigma factor primarily responsible for the acs transcription that cells induce during mid-exponential phase . In contrast, sigma(S) partially inhibits that transcription as cells enter stationary phase. Biol Reprod, 1999 Dec, 61(6), 1649 - 54 Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa; Tsubamoto H et al.; The human zona pellucida (ZP) is composed of three major glycoproteins: ZP1, ZP2, and ZP3 . The aim of this study was to clarify the role of ZP2 by focusing on the polypeptide structure . We produced in Escherichia coli a recombinant human ZP2 protein (rec-hZP2) corresponding to amino acid sequence 1-206 of the mature protein . The final yield of rec-hZP2 protein was 80 microg/ml Luria Broth medium . After 2-h incubation of human spermatozoa with rec-hZP2 in vitro, an immunofluorescent study indicated that rec-hZP2 bound only to acrosome-reacted spermatozoa . The binding site migrated from the acrosome to the midpiece of the spermatozoa . Rabbit and mouse antisera produced against rec-hZP2 stained native human ZP in the immunofluorescent study, and significantly blocked human sperm binding and penetration into human ZP as compared to control values . The N-terminal polypeptide portion of human ZP2 was shown to contain a binding site for acrosome-reacted spermatozoa and to play an important role in secondary sperm binding and penetration into the ZP. Biochemistry, 1999 Sep 21, 38(38), 12229 - 39 Expression, purification, and crystal structure determination of recombinant human epidermal-type fatty acid binding protein; Hohoff C et al.; We describe the crystal structure of human epidermal-type fatty acid binding protein (E-FABP) that was recently found to be highly upregulated in human psoriatic keratinocytes . To characterize E-FABP with respect to ligand-binding properties and tertiary structure, we cloned the respective cDNA, overexpressed the protein in Escherichia coli and purified it to homogeneity by a combination of ion-exchange and size-exclusion chromatographic steps with a yield of 30 mg/L broth . The purified protein revealed a 5-fold higher affinity for stearic acid than for oleic and arachidonic acids . The crystal structure of recombinant human E-FABP was determined to 2.05 A and refined to an R(factor) of 20.7% . The initial residual electron density maps clearly showed the presence of a ligand, which was identified as endogenous bacterial fatty acid . Within a central cavity of 252 A(3), this ligand is bound in a U-shaped conformation, its carboxyl group interacting with tyrosine 131 and arginines 129 and 109, the latter via an ordered water molecule . The E-FABP crystal structure is unique in the FABP family because of the presence of a disulfide bridge between cysteines 120 and 127 that may be physiologically as well as pathophysiologically relevant . Cysteines 67 and 87 are also in close vicinity but in contrast do not form a disulfide bridge . We postulate that this protein belongs to a particular FABP subfamily whose members share common structural as well as functional features. Comp Immunol Microbiol Infect Dis, 1999 Oct, 22(4), 261 - 73 Experimental model of enterotoxigenic Escherichia coli infection in pigs: potential for an early recognition of colibacillosis by monitoring of behavior; Krsnik B et al.; The hypothesis that altered behavior is a sign for an early recognition of disease was tested . The experiment was conducted to evaluate the behavioral patterns of pigs in a model of postweaning colibacillosis . Twenty-five weaned pigs (from a herd that was previously found to be highly susceptible to F4+ Escherichia coli strains) were randomly assigned into 5 groups, kept in isolated pens under the controlled ambiental conditions . One day after weaning, the pigs from three groups were intragastrically inoculated (via orogastric tube) with either F4ac+ (1466 or 2407) or F4- (1467) nonenterotoxigenic E . coli (non-ETEC) strains, respectively . The pigs from the fourth group were inoculated with F4ac+ ETEC strain M1823 and the remaining 5 pigs that received broth containing 1.2% sodium bicarbonate were kept as noninoculated controls . The pigs were examined daily and the frequency and duration of their behavioral patterns, such as eating, drinking, lying, standing, urinating, defecating, rooting and playing were monitored for 300 h during a period of 10 days . In this model, three conditions were also observed in F4-susceptible pigs: (1) acute fatal diarrheal disease; (2) moderate diarrhea and weight loss and (3) no diarrhea and weight loss . The incidence (both frequency and duration) of defecating was significantly higher (P < 0.05) in pigs inoculated with F4ac+ ETEC strain M1823 as compared to that of noninoculated (control) pigs . Pigs inoculated with F4ac+ non-ETEC strain 1466 had a significantly lower frequency of eating (P < 0.05) and frequency/duration of drinking (P < 0.05) than did the controls . The 1466-inoculated pigs, had an increased diarrhea score, but frequency/duration of defecating was not significantly different . Pigs inoculated with F4ac+ non-ETEC strain 2407 spent more time in lying (P < 0.05) than did noninoculated pigs . Conversely, the pigs that received F4- non-ETEC strain 1467 laid shorter (P < 0.05) and ate/drank less frequently (P < 0.05) than the controls . It was concluded that the changed occurrence of defecating and eating in pigs that were inoculated with either F4ac+ ETEC (M1823) or non-ETEC (1466) strain . respectively, was consistent with the pending clinical disease, i.e . postweaning colibacillosis. Appl Environ Microbiol, 1999 Aug, 65(8), 3433 - 40 Heat-induced expression and chemically induced expression of the Escherichia coli stress protein HtpG are affected by the growth environment; Mason CA et al.; Differences in expression of the Escherichia coli stress protein HtpG were found following exposure of exponentially growing cells to heat or chemical shock when cells were grown under different environmental conditions . With an htpG::lacZ reporter system, htpG expression increased in cells grown in a complex medium (Luria-Bertani {LB} broth) following a temperature shock at 45 degrees C . In contrast, no HtpG overexpression was detected in cells grown in a glucose minimal medium, despite a decrease in the growth rate . Similarly, in pyruvate-grown cells there was no heat shock induction of HtpG expression, eliminating the possibility that repression of HtpG in glucose-grown E . coli was due to catabolite repression . When 5 mM phenol was used as a chemical stress agent for cells growing in LB broth, expression of HtpG increased . However, when LB-grown cells were subjected to stress with 10 mM phenol and when both 5 and 10 mM phenol were added to glucose-grown cultures, repression of htpG expression was observed . 2-Chlorophenol stress resulted in overexpression of HtpG when cells were grown in complex medium but repression of HtpG synthesis when cells were grown in glucose . No induction of htpG expression was seen with 2, 4-dichlorophenol in cells grown with either complex medium or glucose . The results suggest that, when a large pool of amino acids and proteins is available, as in complex medium, a much stronger stress response is observed . In contrast, when cells are grown in a simple glucose mineral medium, htpG expression either is unaffected or is even repressed by imposition of a stress condition . The results demonstrate the importance of considering differences in growth environment in order to better understand the nature of the response to an imposed stress condition. Biotechnol Bioeng, 1999 Aug 20, 64(4), 484 - 96 The influence of complex biological feedstock on the fluidization and bed stability in expanded bed adsorption Fernandez-Lahore HM, Kleef R, Kula M, Thommes J. The stability of expanded bed adsorption systems (EBA) was studied in biomass containing culture broth by residence time distribution (RTD) experiments, using pulse inputs of fluorescent molecules as tracers . Different commercial adsorbents (Streamline DEAE, SP, Phenyl, Chelating, and AC) were tested at various biomass concentrations (2.5-12 %, wet weight) of whole (Saccharomyces cerevisiae) yeast, yeast cell homogenate, and Escherichia coli homogenate . Analyzing the RTD according to the PDE model (PDE: axially dispersed plug-flow exchanging mass with stagnant zones) allowed the calculation of three parameters: the number of transfer units for mass exchange between mobile and stagnant fraction (N), the Peclet number for overall axial dispersion (P), and the mobile fraction of the liquid in axially dispersed plug flow (varphi) . When fluidization was performed in particle-free buffer the normalized response signal (after perfect input pulse) was symmetric (N:0; P: 50-100; varphi: 1), thus, demonstrating the formation of a homogeneous fluidized (expanded) bed . Upon application of suspended biomass the RTD was skewed, depending on the adsorbent used and the type and level of biomass present in the sample . This situation leads to three different characteristic pictures: the well-fluidized system (N: >/= 7-10; P: >/= 40; varphi: 0.80-0.90), the system exhibiting bottom channeling (N: < 1-2; P: >/= 40; varphi: 0.5-0.7) and, the system where extensive agglomeration develops (N: 4-7; P: 20-40; varphi: < 0.5) . These results demonstrate that changes in the hydrodynamics of EBA already take place in the presence of moderate concentrations of biomass . Furthermore, those changes can be quantitatively described mainly in terms of the fraction of stagnant zones in the system, which are formed due to the interaction of biomass and adsorbent . The technique described here can be used to evaluate a certain combination of adsorbent and biomass with regard to its suitability for expanded bed adsorption from whole broth . Biosci Biotechnol Biochem, 1999 Apr, 63(4), 672 - 9 Construction of an L-isoleucine overproducing strain of Escherichia coli K-12; Hashiguchi K et al.; The genes for a threonine deaminase that is resistant to feedback inhibition by L-isoleucine and for an active acetohydroxyacid synthase II were introduced by a plasmid into a L-threonine-producing recombinant strain of Escherichia coli K-12 . Analysis of culture broth of the strain using 13C nuclear magnetic resonance suggested that alpha, beta-dihydroxy-beta-methylvalerate (DHMV) and alpha-keto-beta-methylvalerate (KMV), the third and the fourth intermediates in the L-isoleucine biosynthetic pathway from L-threonine, respectively, accumulated in the medium in amounts comparable to that of L-isoleucine . The ratio of accumulated L-isoleucine:DHMV:KMV were approximately 2:1:1 . The concentration of accumulated L-isoleucine increased by twofold after the additional introduction of the genes for dihyroxyacid dehydratase (DH) and transaminase-B (TA-B), and the intermediates no longer accumulated . The resultant strain TVD5 accumulated 10 g/l of L-isoleucine from 40 g/l of glucose. J Bacteriol, 1999 Jun, 181(11), 3525 - 35 Control of acid resistance in Escherichia coli; Castanie-Cornet MP et al.; Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells . Earlier reports described three AR systems in E . coli . In the present study, the genetics and control of these three systems have been more clearly defined . Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS . Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein . The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports . The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:gamma-aminobutyric acid antiporter (gadC) . Only one of the two glutamate decarboxylases was needed for protection at pH 2.5 . However, survival at pH 2 required both glutamate decarboxylase isozymes . Stationary phase and acid pH regulation of the gad genes proved separable . Stationary-phase induction of gadA and gadB required the alternative sigma factor sigmaS encoded by rpoS . However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS independent . Neither gad gene required the presence of volatile fatty acids for induction . The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter . Another surprising finding was that the sigmaS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH . However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction . The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system . Thus, E . coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments. J Biotechnol, 1999 Feb 5, 68(1), 71 - 83 Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli; Schmidt M et al.; The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported . Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin . The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source . The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E . coli resulted in product yields of grams per litre of culture broth, e.g . 4.5 g of insulin B-chain fusion protein per litre of culture broth . This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture . Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture . The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose. Arch Pharm Res, 1998 Jun, 21(3), 305 - 9 Expression of recombinant human cytochrome P450 1A2 in Escherichia coli bacterial mutagenicity tester strain; Chun YJ; Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver . It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines . In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E . coli strain for bacterial reverse mutagenicity assay . Expressed human P450 1A2 showed typical P450 hemoprotein spectra . Maximum expression was achieved at 48 hrs after incubating at 30 degrees C in terrific broth containing ampicillin, IPTG and other supplements . High level expression of P 450 1A2 in E . coli WP2 uvrA membranes was determined in SDS-PAGE . The well-known mutagens 2-aminoanthracene and MelQ increased the revertant colonies of E . coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner . The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals. Mutagenesis, 1998 Nov, 13(6), 589 - 94 Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences; Alhama J et al.; This paper examines the relationship in Escherichia coli between the in vivo content of 8-oxoguanine (8-oxoG) in chromosomal DNA and deficiencies of various key antioxidant defences . The structural genes for catalases (katG and katE), cytosolic superoxide dismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase (fpg) were inactivated to obtain bacterial strains lacking the scavenger enzymes for H2O2 or O2.- or the DNA repair protein for 8-oxoG . Wild-type bacteria showed 5-fold increased sensitivity to both lethality and mutagenesis by H2O2 in K medium (1% casamino acids and 1% glucose), as compared with nutrient broth . This higher sensitivity was associated with increased chromosomal oxidative damage, estimated as the 8-oxodG content, and with a marked decrease in both catalase and SOD activities . Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayed increased 8-oxodG content in chromosomal DNA (2.8-fold that of the wild-type) when grown under standard aerated conditions . Comparatively, no significant difference in 8-oxodG content was observed in cells grown without aeration . Bacteria totally devoid of catalase activity (katG katE mutant) showed wild-type contents of 8-oxodG in chromosomal DNA when grown under aerated conditions . Nevertheless, the protective role of catalase in preventing formation of 8-oxodG in chromosomal DNA became evident under oxidative stress conditions: growth under hyperoxygenation and, particularly, following H2O2 exposure . Catalase deficiency resulted in a dramatic decrease in viability after H2O2 exposure . A deficiency of Fpg protein also sensitized E.coli to H2O2 lethality, though to lesser extent than a deficiency of catalase activity . However, the scavenger enzyme and the DNA repair protein protected equally against 8-oxoG formed in vivo upon H2O2 treatment. Curr Microbiol, 1998 Dec, 37(6), 373 - 9 Effect of IS900 gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis; Naser SA et al.; Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein) . The correlation between the two characteristics has been investigated . A 3.2-kb BamHI fragment from M . paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively . Surprisingly, the recombinant M . smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M . smegmatis wild type or to M . smegmatis transformed with pNEZ6.3 (2 day) . The growth rate of the recombinant M . smegmatis was restored by the addition of 2.4 microM ferric mycobactin J to the media . There was no effect on the growth rate of E . coli recombinants . Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E . coli . There was no expression in the recombinant M . smegmatis . A lower expression of 33 . 5K protein band was detected in the native M . paratuberculosis protein . The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF . There was no additional encoding sequence in the fragment . This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M . paratuberculosis. J Food Prot, 1998 Oct, 61(10), 1312 - 6 Rapid detection and counting of viable bacteria in vegetables and environmental water using a photon-counting TV camera; Miyamoto T et al.; A bioluminescence assay carried out with a photon-counting TV camera was evaluated for rapid enumeration of viable bacterial counts . The test sample was filtered through a membrane filter, and the membrane filter retaining bacteria was incubated at 37 degrees C for 6 h on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl . The membrane filter was then subjected to a bioluminescence reaction, and the intensity of light and numbers of light emission points on the filter were measured with a photon-counting TV camera . The light intensity measured on seven different bacteria correlated with initial viable counts; the correlation coefficient was calculated to be 0.89 . The number of light emission points measured on Escherichia coli also correlated with the initial viable counts (r = 0.81) in a range from 1 to 100 CFU . Presumptive bacterial counts by the present bioluminescence assay determined on 79 samples of vegetables and 122 samples of environmental water correlated well with the viable counts obtained by the conventional plating method, with correlation coefficients of 0.87 and 0.82, respectively. Gastroenterology, 1998 Nov, 115(5), 1123 - 30 Inhibitory effect of somatostatin on Helicobacter pylori proliferation in vitro; Yamashita K et al.; BACKGROUND & AIMS: Somatostatin regulates gastric function and cell proliferation . We investigated whether exogenous somatostatin modulates Helicobacter pylori proliferation in vitro . METHODS: Bacteria were cultured in 5 mL Brucella broth . Bacterial numbers of H . pylori (ATCC 43504) and Escherichia c