Escherichia coli broth

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 coli were calculated 48 and 5 hours after incubation, respectively, by counting the colonies on the blood agar . Chemicals were dissolved in absolute methanol and added to the broth at a final methanol concentration of 1% . Intrabacterial guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) levels were measured by radioimmunoassay . RESULTS: Somatostatin significantly suppressed H . pylori proliferation at levels at or above 10(-11) mol/L . A similar antiproliferative effect was observed with 8-bromo-cGMP . At concentrations at or above 10(-8) mol/L, dibutyryl cAMP slightly but significantly stimulated bacterial proliferation . Gastrin had no effect . Somatostatin antibody immunoglobulin G fraction blocked the antiproliferative effect of somatostatin on ATCC 43504 . Scatchard plot showed that ATCC 43504 has one class of binding site with relatively high affinity (Kd, 0.31 nmol/L) . Somatostatin at 10(-11) mol/L increased cGMP and cAMP in H . pylori 11-fold and 6-fold, respectively . In contrast, somatostatin neither bound E . coli nor affected its proliferation . CONCLUSIONS: Somatostatin, at a similar level in human gastric juice (approximately 10(-11) mol/L), suppresses H . pylori proliferation mediated in part by a cGMP-dependent pathway in vitro, indicating a possible inhibitory effect of somatostatin in the gastric lumen on H . pylori proliferation in humans.

Arch Environ Contam Toxicol, 1998 Nov, 35(4), 588 - 93
Interference by carbohydrate substrates, flavonoids, and monosaccharide derivatives on bacterial beta-D-glucuronidase assays; Mariscal A et al.; Most commercially available test kits for water and foodstuffs use beta-galactosidase activity for coliforms and beta-glucuronidase activity for Escherichia coli . We tested the effects on the beta-glucuronidase activity of E . coli W3110 of substances usually present in foods and several synthetic pharmaceutical compounds . Thirteen substances were tested: three carbohydrates, four flavonoids, five monosaccharide derivatives, and dimethyl sulphoxide . In a minimum medium without any other carbon source, glucose (0.1 mM), quercetin (0.1 mM), silymarin (10 mg/L), D-gluconic acid (0.01 mM), D-gluconic acid lactone (0.01 mM), isopropyl-beta-D-thiogalacto pyranoside (1 mM), p-nitrophenyl beta-D-glucuronide (1 mM), and DMSO (1 M) completely inhibited E . coli glucuronidase activity at the above concentrations . However, the following compounds stimulated E . coli glucuronidase activity within the ranges of concentrations shown: glucose (0.0001-0.01 mM), lactose and sucrose (>0.1 mM), D-saccharic acid 1,4 lactone (0.0001-0.1 mM), p-nitrophenyl beta-D-glucuronide (0.001-0.01 mM) and DMSO (2-500 mM) . In a rich culture medium that contained other carbon sources (lauryl tryptose broth) E . coli glucuronidase activity in the presence of the extra nutrients was unaffected by the test substances and therefore, under normal conditions in water or foods, they should not interfere with E . coli assays based on measurements of beta-glucuronidase activity.

Biotechnol Appl Biochem, 1998 Oct, 28 ( Pt 2), 119 - 24
Renaturation of recombinant human neurotrophin-3 from inclusion bodies using a suppressor agent of aggregation; Suenaga M et al.; Escherichia coli has been widely used in the production of recombinant proteins . One of the drawbacks inherent in this method is that the proteins produced in the cells often form inactive inclusion bodies . Usually, the inclusion bodies can be separated from other cell components, solubilized by denaturants such as guanidine hydrochloride or urea, and then renatured through a refolding process such as dilution or dialysis . However, it has been shown that biologically active recombinant human neurotrophin-3 cannot be obtained at high yield by this procedure due to aggregation and precipitation of the protein . We applied the refolding process using the aggregation suppressor L-arginine in the renaturation of neurotrophin-3, and obtained biologically active neurotrophin-3 at high yield from the inclusion bodies . Consequently, about 10 mg of purified neurotrophin-3 was prepared from 1 litre of culture broth.

J Appl Microbiol, 1998 Sep, 85(3), 615 - 20
Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP; Rowbury RJ et al.; Escherichia coli shifted from broth at external pH (pH0) 7.0 to pH0 7.0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose . Tolerance appeared without the medium pH becoming acidic . Tolerance was most substantial when glucose was added at pH0 7.0 but was also appreciable at pH0 7.5, 8.0 and 8.5 . Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid . Glucose-induced organisms showed less acid damage to DNA and beta-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH0 . In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E . coli at pH0 7.0; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.

Arch Biochem Biophys, 1998 Oct 1, 358(1), 49 - 57
The roles of threonine-136 and glutamate-137 of human medium chain acyl-CoA dehydrogenase in FAD binding and peptide folding using site-directed mutagenesis: creation of an FAD-dependent mutant, T136D; Saijo T et al.; We studied the roles of Thr-136 (T136) and Glu-137 (E137) in the biogenesis of medium chain acyl-CoA dehydrogenase (MCAD) by altering the former to Ser (T136S), Asp (T136D), or Leu (T136L) and the latter to Asp (E137D), Gln (E137Q), or Lys (E137K) . After import into mitochondria, T136S and E137D were assembled into the native tetramer as efficiently as the wild-type . The tetrameric assembly of four other variants with a nonconservative substitution was severely impaired . When expressed in Escherichia coli as the mature subunit, the amounts of the catalytically active forms of T136S and E137D were comparable to wild-type, whereas four nonconservative variants were lost as aggregates . Of these nonconservative variants, only T136D formed catalytically active tetramer when the culture broth and buffers were supplemented with riboflavin and FAD, respectively . Culturing T136L or E137K at a lower temperature (28 degreesC) did not increase the yield at all, suggesting the severity of disruption of biogenesis . These results, together with the previous crystallographic findings, indicate that the T136 hydroxyl is a major FAD-binding site, and that E137 carboxyl plays a key role in the beta-domain folding, through salt bridge formation with K164 . These findings also support the notion that the isoalloxazine ring plays a critical role in the MCAD folding, presumably exerting nucleating effects .

Biotechnol Appl Biochem, 1998 Aug, 28 ( Pt 1), 19 - 23
Investigation of biosynthesis of human lymphotoxin in cells of recombinant Escherichia coli strain; Denisov AA et al.; The effect of cultivation conditions on the biosynthesis of human lymphotoxin in recombinant Escherichia coli SG20050/pLT21 strain was studied . Cells of the producing strain were grown in Luria broth containing chloramphenicol . The highest biomass yield of the recombinant strain and plasmid DNA stability were observed under these conditions . To enhance the level of lymphotoxin production an inoculate containing freshly obtained or frozen with glycerol transformants of the producing strain were used, and the cultivation process was performed at 32 degrees C . As a result, lymphotoxin was synthesized in a soluble form without the formation of inclusion bodies . A study of the protein synthesis dynamics during the cultivation of E . coli at 32 degrees C showed that the highest lymphotoxin activity was observed during the exponential growth phase, being maximal at the end of the exponential phase and at the beginning of the stationary phase . The set of indicated methods allowed us to maximize and stabilize the production of lymphotoxin in a biologically active form with a final yield of 18-20% from cell protein.

Protein Expr Purif, 1998 Aug, 13(3), 423 - 32
Expression, purification, and characterization of Sss1p, an essential component of the yeast Sec61p protein translocation complex; Beswick V et al.; Sss1p, a 8.9-kDa membrane protein, is an essential component of the protein translocation complex involved in the transport of secretory proteins across the Saccharomyces cerevisiae endoplasmic reticulum membrane . In order to determine the high resolution structure of Sss1p by NMR, we have undertaken its overexpression and purification . We first inserted the yeast SSS1 gene into the pGEX-2T plasmid expression vector . Sss1p was expressed as fusions with Schistosoma japonica glutathione S-transferase (GST-Sss1p) in MC1061 Escherichia coli cells . Maximum yield of GST-Sss1p was obtained from cells harvested 2 h after induction at 37 degreesC in Luria broth medium . GST-Sss1p was found associated predominantly with the membrane pool and was readily extracted with Triton X-100 . Detergent-solubilized GST-Sss1p was isolated by adsorption on glutathione-agarose beads . Sss1p was released from its GST carrier by cleavage with thrombin and its recovery was maximized by addition of dodecyl maltoside . Desorbed Sss1p was loaded on a high-performance liquid chromatography hydroxyapatite column equilibrated in phosphate buffer supplemented with dodecyl maltoside and the fractions containing Sss1p were subsequently purified to homogeneity by reverse-phase chromatography on a C4 column . The entire purification protocol can be completed in 5-6 h and yields about 0.4 mg of Sss1p per gram of transformed cells . CD and preliminary 1H NMR experiments show that purified Sss1p solubilized in SDS micelles is very stable and adopts a helical secondary structure .

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 39 - 45
Lethality of high linear energy transfer cosmic radiation to Escherichia coli DNA repair-deficient mutants during the 'SL-J/FMPT' space experiment; Harada K et al.; We investigated the lethal and mutagenic effects of high linear energy transfer cosmic radiation on 11 strains of Escherichia coli, including DNA repair-deficient mutants, using the Radiation Monitoring Container and Dosimeter in the space shuttle 'Endeavour' as part of the 'SL-J/FMPT' space experiment, the 'Fuwatto '92' project . After the return to earth of the shuttle, we evaluated survival and mutations of samples in space and matched controls . The surviving fractions were determined by means of colony count on broth agar plates, and the mutation frequencies were estimated by appearance of arg' revertants on minimal agar plates . The average of the total equivalent dose rate during this space flight was 0.202 mSv/day as measured by the plastic radiation detectors and the thermoluminescent dosimeters in the Radiation Monitoring Container and Dosimeter . The combined action of DNA polymerase and 3'-->5' exonuclease activities was found to make the greatest contribution to the repair of cosmic radiation-induced DNA damage, 5'-->3' exonuclease and recombination repair enzyme activities made a moderate contribution, whereas UV endonuclease activity was not involved in this DNA repair process.

Biomaterials, 1998 Apr-May, 19(7-9), 851 - 9
Bacterial adhesion on PEG modified polyurethane surfaces; Park KD et al.; Polyurethane surface was modified with poly(ethylene glycol) (mol . wt . 1000, PEG1k) carrying terminal hydroxyl, amino and sulfonate groups, poly(ethylene glucol) (mol . wt . 3350, PEG3.4k) and PEG3.4k-Heparin, respectively . These surfaces were investigated for bacterial adhesion using S . epidermidis and E . coli in tryptic soya broth (TSB), brain heart infusion (BHI), and human plasma . All PEG modified surfaces reduced bacterial adhesion significantly and the adhesion level differs depending on surfaces as well as media . In the case of PEG1k surfaces, no reduction of S . epidermidis adhesion was demonstrated in TSB media, regardless of terminal functional groups of PEG1k . However, adhesion in plasma was reduced to the different degree, depending on terminal groups of PEG1k (least adhesion on sulfonated PEG surface) . Relatively longer PEG surface (PEG3.4k) and PEG3.4k-heparin surface minimized bacterial adhesion in both media . In the case of E . coli adhesion, significant reduction in adherent bacteria was observed on all PEG1k, PEG3.4k, and PEG-heparin surfaces in both media compared to controls . In contrast, no reduction in bacterial adhesion was demonstrated on poly(propylene glycol) (PPG1k) grafted PU surface as compared to control PU . These results suggest that surface modification with PEG1k-SO3, PEG3.4k and PEG3.4k-heparin seems to be effective for prevention of bacterial adhesion and subsequent infection.

Appl Microbiol Biotechnol, 1998 Apr, 49(4), 364 - 70
Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli; Shin CS et al.; Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli . The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis . Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E . coli strains . The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone 1(-1) and 7 g fusion glucagon 1(-1) . The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period . The stressful conditions of cultivation employed (i.e., high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins . The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate . The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content.

Poult Sci, 1998 May, 77(5), 654 - 61
The effects of dexamethasone immunosuppression on turkey osteomyelitis complex in an experimental Escherichia coli respiratory infection; Huff GR et al.; Six hundred male turkeys were maintained in floor pens for 5 wk at which time half of the birds were given three intramuscular injections of 2 mg/kg BW of dexamethasone (DEX) on alternating days . On the day of the third DEX injection, the left thoracic air sac of each bird was injected with sterile tryptose phosphate broth (TPB) or with TPB containing approximately 1 x 10(2), 1 x 10(3), 1 x 10(4), or 1 x 10(5) cfu of Escherichia coli . All mortalities and birds necropsied at 14 and 15 d postchallenge were scored for air sacculitis/pericarditis (AS) and turkey osteomyelitis complex (TOC) . Cumulative mortality and AS score were both increased by either DEX treatment or E . coli . Although TOC incidence was significantly increased by the lowest titer of E . coli inoculation, increasing the number of bacteria inoculated did not increase TOC incidence due to increased mortality before TOC lesions developed . The DEX treatment by itself increased TOC incidence and there was a synergistic interaction between DEX treatment and E . coli on TOC incidence . Both DEX treatment and E . coli significantly decreased BW . Relative weights of liver, heart, and spleen were significantly increased by both E . coli and DEX, whereas both treatments significantly decreased relative weight of the bursa of Fabricius . The number of positive bacterial isolations from tissue and the heterophil to lymphocyte ratio were increased by both DEX treatment and E . coli challenge . These results suggest that stress-induced immunosuppression may be involved in the etiology of TOC, and that bacterial respiratory infection can lead to the development of TOC lesions.

Appl Environ Microbiol, 1998 May, 64(5), 1773 - 9
Modelling the growth limits (growth/no growth interface) of Escherichia coli as a function of temperature, pH, lactic acid concentration, and water activity; Presser KA et al.; The form of a previously developed Belehradek type of growth rate model was used to develop a probability model for defining the growth/no growth interface as a function of temperature (10 to 37 degrees C), pH (pH 2.8 to 6.9), lactic acid concentration (0 to 500 mM), and water activity (0.955 to 0.999; NaCl was used as the humectant) . Escherichia coli was unable to grow in broth in which the undissociated lactic acid concentration exceeded 11 mM or, with two exceptions, at a pH of 3.9 or less with no lactic acid present . Under experimental conditions at which the pH and the undissociated acid concentrations were the major growth-limiting factors, the growth/no growth interface was essentially independent of temperature at temperatures ranging from 15 to 37 degrees C . The interface between conditions that allowed growth and conditions at which growth did not occur was abrupt . The inhibitory effect of combinations of water activity and pH varied with temperature . Predictions of the model for the growth/no growth interface were consistent with 95% of the experimental data set.

J Bacteriol, 1998 Apr, 180(8), 2257 - 61
The yhhP gene encoding a small ubiquitous protein is fundamental for normal cell growth of Escherichia coli; Yamashino T et al.; H-NS is a major constituent of the Escherichia coli nucleoid, whereas sigmaS is a stress-induced sigma factor . An hns null mutation affects the cellular content of sigmaS in such a way that a remarkable accumulation of sigmaS is observed in the logarithmic growth phase, which results in enhanced expression of a number of sigmaS-dependent genes, including the katE gene . We isolated an extragenic mutation that affects the expression of the katE-lacZ fusion gene in the deltahns background . The relevant gene was identified as yhhP, which encodes a small polypeptide of 81 amino acids . Lesion of this gene seemed to affect the stability of sigmaS . A deletion analysis of yhhP revealed that this small protein plays a fundamental role in the general physiology of E . coli . The yhhP-deficient cell is not capable of growing in standard laboratory rich medium (i.e., Luria broth), resulting in the formation of filamentous cells . Homologs of this intriguing protein occur in a wide variety of bacterial species, including archaeal species.

Eur J Biochem, 1998 Jan 15, 251(1-2), 462 - 71
Large-scale production, purification and refolding of the full-length cellular prion protein from Syrian golden hamster in Escherichia coli using the glutathione S-transferase-fusion system; Volkel D et al.; Until quite recently, high-level expression of full-length cellular prion protein (Prp(c)) in bacterial cells was not possible . We describe here the effective purification of mature Syrian golden hamster PrPc (residues 23-231) as a C-terminal fusion to glutathione S-transferase (GST) from inclusion bodies expressed in Escherichia coli . Purification of the denatured fusion protein was simplified greatly by the introduction of a C-terminal histidine anchor, leading to 255 mg pure GST-PrPc-His6/l bacterial broth, which could be refolded easily by dilution in 20 mM Tris, 5 mM dithiothreitol, 1 mM EDTA, pH 9.0 . Refolding was monitored by following GST activity . Mature Syrian hamster PrPc (residues 23-231) was released from the refolded fusion protein by thrombin digestion, yielding 73 mg homogeneous protein/l bacterial culture after purification . The recombinant protein was identified by monoclonal antibodies, Edman sequencing and matrix-assisted laser-desorption/ionization MS . Correct folding was confirmed by near-ultraviolet circular dichroism spectroscopy . Samples resulting from different purification steps were sensitive to proteinase K digestion and showed no signs of infectivity in animal experiments, demonstrating that the PrPc produced is identical with the cellular isoform . The presented purification procedure should prove useful for the production of other GST-fusion proteins.

Rev Argent Microbiol, 1997 Oct-Dec, 29(4), 167 - 75
Differential kinetic patterns for Shiga toxin production by Escherichia coli; de Mena MF et al.; We studied the differential kinetic patterns for Shiga toxin (Stx) production (i.e . Stx1, Stx2 and Stx2c) in different reference Escherichia coli strains and in those isolated from hemolytic uremic syndrome (HUS) patients . These results were correlated with those obtained by specific cytotoxic activity assays on Vero cells and hybridization tests with DNA probes for Stx1 and Stx2 . Strains cultured in Penassay broth were sampled at 1.5; 3; 5; 9 and 24 hours to determine bacterial growth and its association with cell-bound and free cytotoxicity . Stx1 showed an intracellular/extracellular concentration ratio (ic/ec) between 32 and 200 times after 3 h-growth . At 24 h both Stx1 concentrations were equal or, in some strains, the ec resulted 2-fold higher that the ic . The ic-Stx1 was equal or just 2-fold higher that ec after 3 h-growth . However, at 24 h the released toxin level was 16 to 32 times higher that cell-bound toxin . The ec-Stx2c increased logarithmically, with maximal yields at 5 h, remaining constant up to 24 h . At that time ic-toxin was 2-fold higher than the released one . When the same experiments were performed on strains isolated from HUS patients they showed that the kinetic patterns obtained corresponded to Stx2 . These results were confirmed by hybridization assays . In this study we have shown that Stx1 production decreases dramatically during stationary phase while Stx2 is detected at high level at that time . This could explain the higher frequency of association of Stx2-producing E . coli strains and HUS in some countries, including Argentina.

Lett Appl Microbiol, 1997 Dec, 25(6), 397 - 400
Effect of centrifugation on the pressure resistance of exponential phase cells of Escherichia coli 8164; Casadei MA et al.; Exponential phase cells of Escherichia coli NCTC 8164 that were centrifuged at 2000 g for 20 min at 4 degrees C were more resistant to subsequent pressure treatment than cells grown in trypticase soya broth (TSB) and treated without any centrifugation steps . The effects of mild pressure stress (200 kPa for 20 min) and temperature stress (a shift from 37 degrees C to 4 degrees C) involved in the centrifugation procedure were analysed separately . It appeared that the increase in pressure resistance obtained following centrifugation was mainly due to the gradual temperature decrease during centrifugation, while the mild pressure stress seemed to play a smaller role in the response.

Infect Immun, 1997 Nov, 65(11), 4572 - 9
Escherichia coli strains with nonimmune immunoglobulin-binding activity; Sandt CH et al.; We have identified several strains of Escherichia coli which contain immunoglobulin-binding activity on the cell surface . Affinity-purified antibodies ordinarily used as secondary antibodies in immunodetection protocols were bound by 6 of 72 strains of the ECOR reference collection of E . coli . The Fc fragments of both human and sheep immunoglobulin G (IgG) were also bound, demonstrating the nonimmune nature of the phenomenon . Binding of conjugated IgG Fc directly to unfixed cells was observed by fluorescence microscopy . Western blots showed that the immunoglobulin-binding material occurs in the form of multiple bands, with the apparent molecular masses of the most prominent bands exceeding 100 kDa . No two of the strains have the same pattern of bands . The binding activity in extracts was sensitive to proteinase K . The binding activity of intact cells was reduced preferentially by trypsin digestion, demonstrating exposure at the cell surface . Expression of binding activity in Luria-Bertani broth cultures was favored by a temperature of 37 degrees C and entry into stationary phase of growth.

Appl Environ Microbiol, 1997 Sep, 63(9), 3526 - 30
A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms; Grant MA; Recovery of total coliforms and Escherichia coli on a new membrane filtration (MF) medium was evaluated with 25 water samples from seven states . Testing of the new medium, m-ColiBlue24 broth, was conducted according to a U.S . Environmental Protection Agency protocol . For comparison, this same protocol was used to measure recovery of total coliforms and E . coli with two standard MF media, m-Endo broth and mTEC broth . E . coli recovery on the new medium was also compared to recovery on nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide . Comparison of specificity, sensitivity, false positive error, undetected target error, and overall agreement indicated E . coli recovery on m-ColiBlue24 was superior to recovery on mTEC for all five parameters . Recovery of total coliforms on the new medium was comparable to recovery on m-Endo.

Arch Microbiol, 1997 Sep, 168(3), 240 - 4
Catabolite regulation of two Escherichia coli operons encoding nitrite reductases: role of the Cra protein; Tyson K et al.; The Escherichia coli nir and nrf operons, which encode alternative nitrite reductases expressed during anaerobic growth, are subject to catabolite regulation . Transcription from the nir promoter is maximal when bacteria are grown in rich media such as Lennox broth supplemented with glucose . Conversely, expression of the nrf operon is suppressed by rich media, but stimulated during growth in minimal medium with glycerol and fumarate . The role of the catabolite repressor-activator (Cra) protein in catabolite regulation of the nir and nrf promoters was investigated . Transcription from the nir promoter was repressed by Cra when cells were grown in minimal medium with glycerol and fumarate . Crude protein extracts from a strain overproducing Cra encoded on a multicopy plasmid retarded a nir promoter fragment in a mobility shift assay, confirming that the observed Cra-dependent repression was due to the direct interaction of Cra with the regulatory region of the nir operon . Furthermore, the inclusion of fructose 1-phosphate, an effector of Cra DNA-binding activity, in the assay decreased the ability of Cra to retard the nir promoter fragment . In contrast, transcription from the nrf promoter was not regulated by Cra under any of the growth conditions tested.

Microbiology, 1997 Aug, 143 ( Pt 8), 2647 - 56
A reassessment of the genetic determinants, the effect of growth conditions and the availability of an electron donor on the nitrosating activity of Escherichia coli K-12; Metheringham R et al.; Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3-diaminonaphthalene when incubated with nitrite . Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate . Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined . Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate . Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth . Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJI operon was implicated as a major contributor to nitrosation activity . Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded . The role of formate in nitrosation was complex and was clearly not limited simply to that of an electron donor in the bacterial reduction of nitrite to nitric oxide: at least two further, chemical roles were inferred . This extensive study of more than 400 independent cultures of E . coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.

J Bacteriol, 1997 Apr, 179(7), 2410 - 7
Cyclic AMP receptor protein functions as a repressor of the osmotically inducible promoter proP P1 in Escherichia coli; Xu J et al.; Transcription of the proP gene, encoding a transporter of the osmoprotectants proline and glycine betaine, is controlled from two promoters, P1 and P2, that respond primarily to osmotic and stationary-phase signals, respectively . The P1 promoter is normally expressed at a very low level under low or normal medium osmolarity . We demonstrate that the binding of the cyclic AMP (cAMP) receptor protein (CRP) to a site centered at -34.5 within the promoter is responsible for the low promoter activity under these conditions . A brief period of reduced CRP binding in early log phase corresponds to a transient burst of P1 transcription upon resumption of growth in Luria-Bertani broth . A CRP binding-site mutation or the absence of a functional crp gene leads to high constitutive expression of P1 . We show that the binding of CRP-cAMP inhibits transcription by purified RNA polymerase in vitro at P1, but this repression is relieved at moderately high potassium glutamate concentrations . Likewise, open-complex formation at P1 in vivo is inhibited by the presence of CRP under low-osmolarity conditions . Because P1 expression can be further induced by osmotic upshifts in a delta crp strain or in the presence of the CRP binding-site mutation, additional controls exist to osmotically regulate P1 expression.

APMIS, 1997 Mar, 105(3), 247 - 54
Expression of colonization factor antigen I fimbriae by enterotoxigenic Escherichia coli; influence of growth conditions and a recombinant positive regulatory gene; Halvorsen T et al.; Enterotoxigenic Escherichia coli (ETEC) may spontaneously lose the positive regulatory cfaR gene and thereby the capacity to express colonization factor antigen I (CFA/I) . A recombinant plasmid harbouring the cfaR gene was transformed into cfaR-negative mutant ETEC strains . CFA/I expression of wild-type and cfaR-transformed ETEC cultivated in different liquid media was quantified . At 37 degrees C, a high level of CFA/I expression from wild-type and cfaR-transformed strains was observed after growth in CFA broth . Transformation enhanced CFA/I expression only marginally . The transformant cultures showed a considerable variation in CFA/I expression which was paralleled by the proportion of individual bacteria producing CFA/I . This heterogeneity could be explained by a variable tendency to structural CFA/I gene loss among individual cfaR-transformed bacteria.

Vet Rec, 1997 Feb 1, 140(5), 112 - 5
Studies on the development and use of a monoclonal sandwich ELISA for the detection of verotoxic Escherichia coli in animal faeces; Randall LP et al.; A sandwich ELISA using monoclonal antibodies to Escherichia coli verocytotoxins 1 and 2 for capture and detection was developed for detection of verocytotoxin-producing Escherichia coli (VTEC) in animal faeces . For optimal toxin detection, the faeces were cultured in MacConkey broth before subculture on to trypticase soy agar containing mitomycin C . It was possible to detect between 10 and 10(3) VTI-producing and 1 to 10 VT2-producing E coli/g faeces, and overall the sensitivity and specificity of the ELISA for VTEC in faeces were 80.5 per cent and 91.2 per cent, respectively, when compared with a verocell assay . A limited survey of faeces from healthy animals (89 cattle, 16 sheep, 22 pigs, 11 goats and six domestic fowl) and faeces from 144 cattle with enteric disease detected VTEC in 64 per cent, 63 per cent, 5 per cent, 45 per cent, none and 16 per cent of the samples, respectively.

Lett Appl Microbiol, 1996 Oct, 23(4), 269 - 72
Induction of the PhoE porin by NaCl as the basis for salt-induced acid sensitivity in Escherichia coli; Lazim Z et al.; Organisms grown in low salt broth (LSB) are acid resistant but become sensitive on growth for 30-60 min with 300 mmol l-1 added NaCl . Salt-induced acid sensitivity only occurs in relA+ strains and sensitization is abolished by glucose, this catabolite repression effect being reversed by cAMP . The finding that sensitization did not occur in a phoE strain but did occur in a phoE+ derivative of it suggested that the response might result from PhoE induction, since PhoE acts as the major outer membrane (OM) proton pore under most conditions . In agreement with this, low-salt broth (LSB)-grown cells of a chromosomally lac- strain carrying pJP102 (phoE-lacZ) produced low levels of beta-galactosidase but growth with added NaCl led to rapid and appreciable induction . Also, a phoA mutant carrying a phoE-phoA fusion produced little alkaline phosphatase after growth in LSB but much more in LSB with added NaCl . Increased beta-galactosidase synthesis (in phoE-lacZ strains) in the presence of NaCl was abolished by glucose, this effect being reversible by cAMP, and there was more NaCl-induced synthesis of this enzyme in relA+ strains . Accordingly, it appears that addition of NaCl to LSB leads to acid sensitivity because it induces synthesis of the OM proton pore PhoE.

Microbiologia, 1996 Sep, 12(3), 395 - 404
Evaluation of an enzyme immunoassay for verotoxin detection in Escherichia coli; Frias C et al.; Verotoxin-producing Escherichia coli strains (VTEC) cause hemorrhagic colitis and hemolytic-uremic syndrome in humans . Laboratory diagnosis by conventional methods is slow and cumbersome . The results of a new rapid enzyme immunoassay (EIA Premier EHEC) for verotoxin detection both in isolated strains and in clinical samples are presented, and they are compared with cell culture (CC) and polymerase chain reaction (PCR) techniques . Fifty-four strains have been analyzed by both EIA and PCR, and 33 by all three methods . The kit has also been evaluated for experimentally infected stool samples directly and after their enrichment on MacConkey broth . Nineteen, out of the 54 strains, were positive by EIA and 20 by PCR . The results of the 33 strains evaluated by the three techniques were coincident with one exception . The latter was uninterpretable by CC, negative by EIA and positive by PCR . The sensitivity of the kit for experimentally infected stool samples was approximately 5 x 10(7) bacteria/ml in the direct test, and 5 x 10(4) bacteria/ml after broth enrichment . EIA sensitivity and specificity were similar to those of CC and PCR . The diagnostic times were 18h for EIA, 3 days for PCR and 5 days for CC . Sensitivity, rapidity and ease of performance make this technique especially valuable for clinical diagnosis.

Mol Microbiol, 1996 Sep, 21(5), 953 - 61
Activation of stable DNA replication in rapidly growing Escherichia coli at the time of entry to stationary phase; Hong X et al.; The conditions are described in which DNA replication can occur, in the absence of protein synthesis, in wild-type Escherichia coli cells . Chromosome replication, which is normally inhibited by addition of chloramphenicol, becomes resistant to this drug after nutritional shiftup, e.g . from minimal medium to Luria broth . This replication activity appears transiently when nutritionally upshifted cells enter stationary phase . The activity strictly requires recA+, but it is independent of recB+ and dnaA+ . It can occur in the absence of concomitant transcription . Activation of the replication does not result from induction of the SOS response . As the characteristics of this DNA replication resemble those of the previously characterized stable DNA replication, it is termed nutritional shiftup-activatable stable DNA replication, nSDR . Possible mechanisms of the activation of nSDR in rapidly growing cells at the time of entry to stationary phase are discussed.

J Bacteriol, 1996 Sep, 178(18), 5487 - 92
Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12; Poole RK et al.; Escherichia coli possesses a soluble flavohemoglobin, with an unknown function, encoded by the hmp gene . A monolysogen containing an hmp-lacZ operon fusion was constructed to determine how the hmp promoter is regulated in response to heme ligands (O2, NO) or the presence of anaerobically utilized electron acceptors (nitrate, nitrite) . Expression of the phi (hmp-lacZ)1 fusion was similar during aerobic growth in minimal medium containing glucose, glycerol, maltose, or sorbitol as a carbon source . Mutations in cya (encoding adenylate cyclase) or changes in medium pH between 5 and 9 were without effect on aerobic expression . Levels of aerobic and anaerobic expression in glucose-containing minimal media were similar; both were unaffected by an arcA mutation . Anaerobic, but not aerobic, expression of phi (hmp-lacZ)1 was stimulated three- to four-fold by an fnr mutation; an apparent Fnr-binding site is present in the hmp promoter . Iron depletion of rich broth medium by the chelator 2'2'-dipyridyl (0.1 mM) enhanced hmp expression 40-fold under anaerobic conditions, tentatively attributed to effects on Fnr . At a higher chelator concentration (0.4 mM), hmp expression was also stimulated aerobically . Anaerobic expression was stimulated 6-fold by the presence of nitrate and 25-fold by the presence of nitrite . Induction by nitrate or nitrite was unaffected by narL and/or narP mutations, demonstrating regulation of hmp by these ions via mechanisms alternative to those implicated in the regulation of other respiratory genes . Nitric oxide (10 to 20 microM) stimulated aerobic phi (hmp-lacZ)1 activity by up to 19-fold; soxS and soxR mutations only slightly reduced the NO effect . We conclude that hmp expression is negatively regulated by Fnr under anaerobic conditions and that additional regulatory mechanisms are involved in the responses to oxygen, nitrogen compounds, and iron availability . Hmp is implicated in reactions with small nitrogen compounds.

Int J Food Microbiol, 1996 Jul, 30(3), 217 - 29
Enterotoxigenic Escherichia coli detected in foods by PCR and an enzyme-linked oligonucleotide probe; Deng MY et al.; A polymerase chain reaction (PCR) and an enzyme-linked oligonucleotide probe hybridization assay were developed for the detection of enterotoxigenic Escherichia coli (ETEC) in ground beef, chicken, pork and raw milk . Two synthetic primers, one of which was biotinylated, were used in the PCR to amplify a fragment of the E . coli heat-labile enterotoxin (LT) gene . The identity of the amplified products was confirmed by liquid hybridization using a horseradish peroxidase-linked internal oligonucleotide probe in a 96-well microplate coated with streptavidin . The final quantitation of the PCR products was performed by a colorimetric reaction . Under established conditions (including 1 min at 60 degrees C for primer annealing and extension in PCR cycles), this method detected all 7 LT-producing E . coli pathogenic for humans, but did not detect all 7 LT-positive E . coli of animal origin 3 E . coli strains that do not produce LT, and 9 other bacteria . Under less stringent PCR conditions (55 degrees C for annealing and extension), 2 strains of LT-producing E . coli of porcine origin were detected while the results of other bacterial strains remained unchanged . In pure cultures, the detection limit of the method was 1.4 colony forming units (CFU) . Prior to PCR amplification, all food samples inoculated with an LT-producing ETEC, were subjected to enrichment in brain heart infusion broth for 8 h at 37 degrees C . From these cultures, 10 microliters was heated at 95 degrees C for 10 min and directly used in the PCR . An initial inoculum of as few as 1.2 to 12 CFU of the LT-producing ETEC per 25 g (or ml) of food sample gave a positive reaction.

Enferm Infecc Microbiol Clin, 1996 May, 14(5), 308 - 10
{Errors of the agar diffusion method to predict Escherichia coli's susceptibility to ampicillin-sulbactam and amoxicillin-clavulanic acid}; Villar HE et al.; BACKGROUND: To evaluate the in vitro activity of the ampicillin-sulbactam and amoxicillin-clavulanic acid against Escherichia coli isolations resistant to ampicillin and amoxicillin and the efficacy of the disks of ampicillin sulbactam 10/10 microgram and amoxicillin-clavulanic acid 20/10 micrograms to differentiate the susceptible (S) and resistant (R) isolates . METHODS: We evaluated the in vitro susceptibility of 100 consecutive clinical isolates of ampicillin and amoxicillin resistant E . coli by the broth macrodilution method and disk diffusion test against ampicillin-sulbactam and amoxicillin-clavulanic acid . RESULTS: For amoxicillin-clavulanic acid the 64% of the isolates were susceptible, 34% were moderately susceptible and 2% were resistant . In contrast, the in vitro activity of ampicillin-sulbactam was inferior since 13% of the isolates were susceptible, 24% moderately susceptible and 63% were resistant . By using the disk of ampicillin-sulbactam 10/10 microgram we found a 13% of very major errors and a 44% of minor errors when we consider the actual rules of NCCLS (R < or = 11 mm and S > or = 15 mm) . The best results were achieved when we took into account zone size < or = 15 mm as R and > or = 20 mm as S; however, the level of errors was high too (25% minor errors) . For the disk of amoxicillin-clavulanic acid 20/10 micrograms we found a 31% of minor errors when using the advised break points (R < or = 13 mm and S > or = 18 mm) . CONCLUSIONS: We consider that the disk diffusion tests are not applicable to these combinations when E . coli isolates resistant to aminopenicillin are evaluated . We advise not to extrapolate the results of sensibility or resistance from one combination to the other because it presents a different in vitro activity.

Biotechniques, 1996 May, 20(5), 854 - 6, 858, 860
Improved method for the production of M13 phage and single-stranded DNA for DNA sequencing; Reddy P et al.; An improved method is described for the efficient production of M13 phage and M13 single-stranded (ss)DNA in a relatively short time period . Infection of E . coli (F') cells with as few as 5 phage particles can yield 10(12) phage particles/mL in 3 hours if the cells are grown in LB broth or SOB broth supplemented with about 5 mM Mg2+ . The method tolerates large variations in the initial multiplicity of infection (5-5000 phage per 5 x 10(7) cells) and still yields about 10(12) phage particles/mL . These amounts are sufficient to purify 10-15 micrograms of ssDNA and to carry out at least 10-15 DNA sequencing reactions.

Appl Environ Microbiol, 1996 Apr, 62(4), 1347 - 53
A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef; Witham PK et al.; A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes . This quantitative detection system involves the 5'-->3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein {FAM}) and a quencher dye (6-carboxytetramethyl-rhodamine {TAMRA}) . Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology . The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer . When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, delta RQ values increased from approximately 1.5 to 3.0 . The delta RQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM . The assay is sensitive and can detect approximately 10 +/- 5 CFU per PCR . As few as 0.5 CFU of Shiga-like toxin I-producing E . coli per g could be detected in ground beef with only 12 h of enrichment in modified E . coli broth.

J Bacteriol, 1996 Mar, 178(5), 1258 - 64
The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair; Kogoma T et al.; The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication . Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks . The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication . Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants . The spa-47 suppressor mutation maps within or very near dnaC . These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E . coli involve extensive DNA replication.

J Bacteriol, 1996 Feb, 178(3), 668 - 74
A regulator of the flagellar regulon of Escherichia coli, flhD, also affects cell division; Pruss BM et al.; The role of an activator of flagellar transcription in Escherichia coli, flhD, was investigated in the regulation of cell division . When grown in tryptone broth, flhD mutant cells divided exponentially until they reached a cell density of 2.5 x 10(9) cells per ml . Wild-type cells and flhC mutant cells divided exponentially until they reached a cell density of 4 x 10(7) cells per ml . flhD mutant cells divided 5 times more than wild-type cells before they reduced their cell division rate and reached a cell density 37 times higher than that of wild-type or flhC mutant cultures . In stationary phase, the biomasses of all cultures were similar; however, flhD mutant cells were significantly smaller . Additional tryptone, Casamino Acids, and individual amino acids, added at the beginning of growth, allowed wild-type cells to grow to higher cell densities . Serine was determined to have the greatest effect . In contrast, the addition of Casamino Acids did not exhibit an effect upon flhD mutant cells . flhD mutant cells exhibited normal rates of uptake of serine and other amino acids . In both wild-type and flhD mutant cultures, the concentrations of serine in the media dropped from 140 to 20 microM within the first 2 h of growth . Serine concentrations and cell division rates were highly correlated . Wild-type cells reduced their cell division rate at a medium concentration of 50 microM serine, and the addition of serine at this time caused cells to resume a higher rate of division . We conclude that the reduction of the cell division rate in wild-type cells is caused by the depletion of serine from the medium and that flhD mutant cells seem to be unable to sense this depletion.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 459 - 65
Optimization of bacteriocin-release-protein-induced protein release by Escherichia coli: extracellular production of the periplasmic molecular chaperone FaeE; van der Wal FJ et al.; Expression of the pCloDF13-encoded bacteriocin-release protein (BRP) results in the release of periplasmic proteins into the culture medium . The BRP-mediated release of a periplasmic protein was investigated and optimized . As a periplasmic model protein, the 50-kDa dimeric E., coli fimbrial molecular chaperone FaeE was used . Plasmids were constructed for the simultaneous expression of the BRP and FaeE, controlled by independently inducible promoters . The efficiency of FaeE release increased when the BRP was targeted by the unstable murein lipoprotein signal peptide, instead of by its own stable signal, peptide . Furthermore, optimal efficacy of FaeE release was found when cells of E . coli strain C600 were used, which harboured one plasmid encoding both FaeE and BRP instead of two separate plasmids and which were cultured at 37 degrees C in broth supplemented with MgCl2 . Maximal production levels of 21 mg FaeE/l culture were obtained.

Mol Gen Genet, 1995 Sep 20, 248(5), 629 - 34
An Escherichia coli curved DNA-binding protein whose expression is affected by the stationary phase-specific sigma factor sigma S; Kakeda M et al.; From Escherichia coli, a DNA-binding protein that preferentially recognizes a curved DNA sequence was isolated and shown to correspond to one that has recently been reported as a binding protein for the replication origin of the E . coli chromosome, named Rob . Here, a rob promoter-lacZ transcriptional fusion was constructed on the chromosome, and used to demonstrate that the expression of rob is notably enhanced at the onset of stationary phase in Luria-broth and also under certain growth conditions in a minimal medium, such as glucose- and phosphate-starvation medium . It was further shown that this growth condition-dependent expression of rob is notably reduced in a null mutant for the stationary phase-specific sigma subunit of RNA polymerase, sigma s, although sigma s-independent expression of rob was significant during the logarithmic growth phase . Furthermore the rob null mutant was found to exhibit, as compared with the wild-type, an altered profile of protein synthesis, particularly at the very late stationary phase.

J Bacteriol, 1995 Sep, 177(17), 4851 - 6
Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli; Maupin-Furlow JA et al.; DNA sequence analysis of the modABCD operon of Escherichia coli revealed the presence of four open reading frames . The first gene, modA, codes for a 257-amino-acid periplasmic binding protein enunciated by the presence of a signal peptide-like sequence . The second gene (modB) encodes a 229-amino-acid protein with a potential membrane location, while the 352-amino-acid ModC protein (modC product) contains a nucleotide-binding motif . On the basis of sequence similarities with proteins from other transport systems and molybdate transport proteins from other organisms, these three proteins are proposed to constitute the molybdate transport system . The fourth open reading frame (modD) encodes a 231-amino-acid protein of unknown function . Plasmids containing different mod genes were used to map several molybdate-suppressible chlorate-resistant mutants; interestingly, none of the 40 mutants tested had a mutation in the modD gene . About 35% of these chlorate-resistant mutants were not complemented by mod operon DNA . These mutants, designated mol, contained mutations at unknown chromosomal location(s) and produced formate hydrogenlyase activity only when cultured in molybdate-supplemented glucose-minimal medium, not in L broth . This group of mol mutants constitutes a new class of molybdate utilization mutants distinct from other known mutants in molybdate metabolism . These results show that molybdate, after transport into cells by the ModABC proteins, is metabolized (activated?) by the products of the mol gene(s).

Vet Microbiol, 1995 Aug, 45(4), 297 - 309
Expression of P and type 1 (F1) fimbriae in pathogenic Escherichia coli from poultry; Dozois CM et al.; To investigate the expression of P and type 1 (F1) fimbriae in pathogenic avian Escherichia coli, fourteen pap+/fim+ E . coli isolates pathogenic for poultry were grown on four complex or minimal media, and examined for the presence of mannose resistant (MR) and mannose sensitive (MS) hemagglutination (HA), and for P or for type 1 (F1) fimbriae using immunofluorescence, immunodot, and immunoblot . In addition, isolates grown under different culture conditions were examined for adherence to frozen sections of chicken trachea . Twelve of the 14 isolates were divided into three groups based on adhesin expression in the different media . Isolates of all three groups exhibited strong MSHA reactions when cultures were grown serially in static broth, and expressed a subunit protein with an apparent molecular mass of 17 to 18.5 kDa, serologically related to the FIA major fimbrial subunit . There was a good correlation between MSHA and adherence to chicken tracheal sections . Isolates of group I only demonstrated MSHA and expression of F1A fimbriae after growth in static broth . Isolates of group II demonstrated MSHA and expression of F1A fimbriae after growth in all tested media whereas isolates of group III demonstrated expression of F1A fimbriae only after growth in static broth and minimal agar . Only the five group I isolates expressed MRHA associated with P fimbrial adhesins and expressed fimbriae with a major subunit protein of 18 kDa serologically related to the F11 major fimbrial subunit . None of these five isolates grown on complex solid media, where P but not type 1 fimbriae were expressed, adhered to tracheal sections . Results suggest that i) P fimbriae are not readily expressed in vitro by most pap+/fim+ avian E . coli isolates; ii) environmental control of phase variation of type 1 fimbriae differs among pathogenic avian E . coli; and iii) receptors for type 1, but not for P fimbriae, are present in chicken tracheal mucosa.

Microbiology, 1995 Jul, 141 ( Pt 7), 1621 - 7
Production of Escherichia coli STb enterotoxin is subject to catabolite repression; Busque P et al.; Enterotoxigenic Escherichia coli are known to secrete several types of toxins including STb, a heat-stable enterotoxin . STb enterotoxin production was studied in wild-type E . coli strains . Using a quantitative STb-specific inhibition ELISA, the amount of toxin present in the culture supernatant fractions of various E . coli strains was determined . Variation in the production of STb toxin was observed for the wild-type strains . For E . coli strain 82-4247 grown in trypticase soy broth, the toxin was produced after 4 h of growth and was maximal after about 57 h of growth . The amount of toxin in the culture supernatant fraction increased concomitantly with bacterial growth . Using the rat loop assay, the biological activity of STb was retained even after the logarithmic phase of growth when STb production levelled off (i.e . from 24 to 74 h) . STb production by E . coli strain 82-4247 varied with the culture medium used . In particular, addition of 1.0% (w/v) compared to 0.1% glucose to Davis minimal medium decreased STb production, whereas addition of 1.0% (w/v) glycerol did not affect STb production . Addition of exogenous cAMP reversed the repressive effect of glucose . Using mutant strains, STb production was shown to be subject to catabolite repression.

J Clin Microbiol, 1995 May, 33(5), 1414 - 7
Escherichia coli adherence to HEp-2 cells with prefixed cells; Zepeda-Lopez HM et al.; We describe a new method which uses cold absolute methanol-prefixed cells for adherence of enteropathogenic Escherichia coli to HEp-2 cells . We found that a method using bacteria grown in Penassay broth to 10(6) to 10(7) CFU/ml and incubated with prefixed cells for 3 h at 37 degrees C, showed 100% sensitivity and specificity against a method using live cells.

J Antimicrob Chemother, 1995 May, 35(5), 603 - 9
Positive R plasmid mutator effect on chromosomal mutation to nalidixic acid resistance in nalidixic acid-exposed cultures of Escherichia coli; Ambler JE et al.; Mutation frequencies to nalidixic acid resistance (15 mg/L in nutrient agar) were determined for derivatives of Escherichia coli AB1157 carrying the mutator plasmids R46, R391 or pYD1, or the non-mutator plasmid RP4 . Frequencies of mutation remained constant in cultures of AB1157(R46) growing exponentially in drug-free broth, at a level about 12-fold higher than in the strain without plasmid . Mutation frequencies in cultures of strains AB1157(R391) and AB1157(pYD1) were about three times greater than in the control, whereas plasmid RP4 had no effect on spontaneous mutation frequency to nalidixic acid resistance . Exposure of strain AB1157 to 6 mg/L nalidixic acid in nutrient broth killed 80% of cells after 4 h . This enriched the proportion of nalidixic acid-resistant cells present in the surviving cell population giving enhanced "apparent" mutation frequencies . These were further increased by cell division of resistant mutants in the nalidixic acid-containing medium . "Apparent" resistance mutation frequencies in nalidixic acid-exposed cultures of the R46-, R391- or pYD1-carrying derivatives were, at their peak, 447-, 53- and 38-fold higher than in the control, the strain without plasmid, or the RP4-containing strain, respectively . These data illustrate how mutator plasmids like R391 and pYD1, which mediate only small increases in spontaneous mutation, can contribute to the development of clinically-significant levels of quinolone resistance.

Eur J Biochem, 1995 Apr 15, 229(2), 533 - 9
The preparation of catalytically active human cathepsin B from its precursor expressed in Escherichia coli in the form of inclusion bodies; Kuhelj R et al.; A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies) . The recombinant product was solubilized and renatured by refolding and reoxidation . The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme . By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/l E . coli culture broth . The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three-amino-acid extension at its N-terminus . The enzyme has similar kinetic and immunological properties to native human cathepsin B.

J Bacteriol, 1995 Apr, 177(8), 2209 - 13
A stationary-phase-dependent viability block governed by two different polypeptides from the RhsA genetic element of Escherichia coli K-12; Vlazny DA et al.; Multicopy plasmids bearing a small internal portion of the RhsA genetic element of Escherichia coli K-12 imparted a viability block on cultures grown to stationary phase in broth . Inclusion of the last 25 codons of the RhsA core open reading frame (called core-ORF) in the plasmid insert was crucial for eliciting this toxic effect . The toxic effect could be suppressed by including the adjacent Rhs component, dsORF-a1, on the multicopy plasmid . The toxic effect was enhanced in RpoS- strains.

Mol Microbiol, 1995 Apr, 16(1), 97 - 109
Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions; Mobley HL et al.; Urease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastritis and peptic ulceration . This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea . When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structural subunits are overexpressed, exogenous NiCl2 is added, and the host strain is grown in medium that does not chelate free Ni2+ . As wild-type H . pylori does not require such conditions for very high levels of urease expression, we reasoned that additional genes were required to accumulate the metal ion . To isolate such genes, E . coli SE5000 (pHP808), which carries the H . pylori urease gene cluster, was complemented with a lambda ZAP-derived plasmid library of the H . pylori chromosome . One of 1000 ampicillin-resistant clones, plated onto urea segregation agar, produced detectable urease . Urease activity of this co-transformant, grown in Luria broth containing 1 microM NiCl2, was 36 mumol NH3 min-1 mg-1 protein . Urease-enhancing activity, which is not directly linked to the urease gene cluster, was localized by subcloning and nucleotide sequencing . The largest open reading frame, designated nixA, predicted a polypeptide of 34,317 Da that displayed characteristics of an integral membrane protein . In vitro transcription-translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size . An in-frame Bal31 deletion within nixA abolished urease-enhancing activity . At 50 nM NiCl2, E . coli containing the nixA clone transported 1250 +/- 460 pmol Ni2+ min-1 10(-8) cells, whereas the vector control transported only 140 +/- 85 pmol Ni2+ min-1 10(8) cells, i.e . significantly less (P = 0.01) . We conclude that NixA confers upon E . coli a high-affinity nickel-transport system (KT = 11.3 +/- 2.4 nM; Vmax = 1750 +/- 220 pmol Ni2+ min-1 10(-8) cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.

Biosci Biotechnol Biochem, 1995 Feb, 59(2), 256 - 61
Decreasing accumulation of acetate in a rich medium by Escherichia coli on introduction of genes on a multicopy plasmid; Hosono K et al.; Escherichia coli excretes acetate during aerobic growth in a rich medium, L-broth containing 0.4% glucose, and growth ceases before depletion of glucose because of the decrease in pH caused by the accumulation of acetate . The addition of sodium phosphate buffer to the medium allows cells to reuse the acetate accumulated . Reuse of the acetate, however, does not occur in the presence of remaining glucose . A gene on a multicopy plasmid was found to significantly decrease the accumulation of acetate by the transformant and the growth did not cease until depletion of both the glucose and acetate in the medium . The gene was tentatively named mlc (making large colonies) . The putative Mlc protein has high hology with the NagC protein, which is a regulator protein in the nag operon responsible for the use of N-acetylglucosamine . The nagC gene on a multicopy plasmid also decreased the accumulation of acetate . Although the function of the genes in the phenomenon described is still unclear, transformants harboring the mlc gene or nagC gene on a multicopy plasmid will be useful for condensed cultivations involving glucose.

Vet Microbiol, 1995 Feb, 43(2-3), 183 - 96
Partial characterization of a Moraxella bovis cytolysin; Gray JT et al.; Moraxella bovis (M . bovis) is the etiologic agent of infectious bovine keratoconjunctivitis and M . bovis hemolysin is believed to be an important virulence factor . Two strains of M . bovis were compared, Epp 63(300) (Epp), a known virulent and hemolytic strain, and IBH 63 (IBH), a known avirulent and nonhemolytic strain . Sterile 10-fold (10x) supernatant concentrates were obtained from cultures grown in TSB broth with 10 mM CaCl2 . Supernatant hemolysin titers for Epp, were 1:1024 and 1:8192 for unconcentrated (1x) and 10x, respectively . Supernatant cytotoxin titers to bovine mononuclear cells were 1:32 and 1:128 for 1x and 10x, respectively, for Epp . Cytolytic (hemolytic and cytotoxic) activities declined 10-fold but were still measurable for > 1 wk at 4 degrees C . Both activities were inactivated by trypsin and by heating at 56 degrees C for 20 min . A cytotoxic effect was observed on cultured bovine and ovine corneal epithelial cells with Epp . All cytolytic effects were neutralized with antiserum to 10x Epp . No cytolytic activities were detected for 10x IBH . SDS-PAGE electrophoresis and related immunoblots indicate a high molecular weight protein at 110 kDa for the 10x Epp preparation when stained with silver or probed with monoclonal antibodies to the E . coli alpha hemolysin . No 110 kDa band is observed for 10x IBH . These data suggest that hemolytic and cytotoxic activities are important in the pathogenesis of infectious bovine keratoconjunctivitis and identify the protein as a possible RTX related toxin of 110 kDa . Stability of the M . bovis cytolysin for > 1 week should allow further characterization and purification of the protein.

Mikrobiol Z, 1995 Jan-Feb, 57(1), 44 - 7
{The reaction of Escherichia coli cells to an acid nonnutritive medium}; Dyl'ovyi MV; Cells of Escherichia coli K 12 grown on nutritious broth and twice washed off from medium by 90 mM solution of lithium chloride alkalize to some degree acidic non-nutritional medium . Registration of rates of alkalization by the cells at constant pH (from 3.0 to 5.5) was made by means of automatic potentiometric titration . The initial rate of alkalization was higher at low pH of the incubation medium . Different mechanisms of medium alkalization by bacterial cells (consumption of protons by cells, antiport proton/metal ion and release of basic products by cells) are discussed.

Arch Virol, 1995, 140(10), 1817 - 31
Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein; Smith GR et al.; A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E . coli . The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site . The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region . The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT) . Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides . This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein . This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa . Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis . The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea . A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth . The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E . coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

J Bacteriol, 1994 Dec, 176(24), 7727 - 9
Activity of the Escherichia coli mutT mutator allele in an anaerobic environment; Fowler RG et al.; Mutation frequencies for an Escherichia coli mutT strain were measured in both aerobic and anaerobic environments . When cells were grown in a rich medium (L broth), mutation frequencies were similar in both aerobic and anaerobic conditions . In contrast, when grown in a minimal medium, mutT anaerobic mutation frequencies were reduced dramatically compared with aerobic values, which were similar to L broth frequencies . L broth mutT cultures treated with a commercial enzyme complex that reduces free oxygen in the medium also showed strongly reduced anaerobic mutation frequencies . These results indicate that the biological role of the MutT protein is to prevent oxidative damage from becoming mutagenic.

J Chem Technol Biotechnol, 1994 Nov, 61(3), 273 - 81
Efficient fuzzy control strategies for the application of pH-stat to fed-batch cultivation of genetically engineered Escherichia coli; Jin S et al.; In the cultivation of genetically engineered Escherichia coli it is very important to control the substrate concentration at an appropriate level in order to avoid the accumulation of acetate, thereby elevating the expression level of plasmid-encoded protein . In this paper, a pH-stat mode of fuzzy control was considered for the overexpression of beta-galactosidase in the fed-batch cultivation of recombinant E . coli . In the simple pH-stat fuzzy control, the response of pH change in the culture broth to the feeding rate of glucose was used to estimate the glucose consumption rate . In the modified pH-stat fuzzy control, the glucose consumption rate was accurately estimated by using pH change and the change in the carbon dioxide content of the exhaust gas . With this control strategy, the cell density could be increased to 72 g DCW dm-3, which was twofold higher than that attained in the cultivation with the simple pH-stat fuzzy control . The bulk beta-galactosidase concentration was increased to 4150 U cm-3, which was threefold higher than when the simple pH-stat control was used.

Zh Mikrobiol Epidemiol Immunobiol, 1994 Sep-Oct, (5), 6 - 10
{The development of nutrient media for the cultivation of recombinant Escherichia coli strains}; Santsevich NI et al.; The study of the qualitative and quantitative composition of peptides and free amino acids in commercial casein hydrolysates, produced in Russia, has provided grounds for the choice of the nutrient base in the medium for the cultivation of E . coli recombinant strains . In the newly developed medium specific endoglucanase activity is 1.5- to 1.7-fold greater than in Luria-Bertani broth ("Difco" Laboratories, USA).

J Photochem Photobiol B, 1994 Aug, 24(3), 155 - 61
Two different mechanisms of low-intensity laser photobiological effects on Escherichia coli; Karu T et al.; Bacterial suspensions in a phosphate buffer were irradiated at wavelengths lambda of 632.8, 1066 and 1286 nm, incubated in Hottinguer broth for 60 min and assayed for viability by the standard surface-plating technique . The difference between the number of viable cells in the irradiated culture and the control was termed growth stimulation . Irradiation of the bacteria with an He-Ne laser (632.8 nm) or semiconductor lasers at 1066 and 1286 nm at various intensities and irradiation times produced two maxima in the growth stimulation vs . dose curve . The first maximum, in all cases, occurred near 50 J m-2, and the reciprocity law was obeyed . The second maximum occurred at an irradiation time of 100 s irrespective of the particular radiation intensity, and the reciprocity law was not obeyed . It is assumed that two different mechanisms are responsible for these two maxima in the growth stimulation vs . dose curve.

Appl Microbiol Biotechnol, 1994 Aug, 41(6), 677 - 83
High-yield production of human big endothelin-1 by a combination of chemical modification and proteolysis of a fusion protein in Escherichia coli; Ohashi H et al.; A protein modification method has been developed for the production of human big endothelin (ET)-1 . Production of a large quantity of big ET-1 by the method described here is expected to facilitate future experiments such as X-ray crystallography and nuclear magnetic resonance studies, aimed at understanding the tertiary structure of big ET-1 and its dynamics . The plasmid pETB-50 used for the synthesis carries the gene for a fusion protein consisting of 34-amino acid (aa) residues of an N-terminal portion of beta-galactosidase and the 38-aa residues of big ET-1 . The fusion protein ETB-50P contains an arginine residue in the big ET-1 portion at its second C-terminal site and three lysine residues including the C-terminal site in the beta-galactosidase portion, all of which are susceptible to trypsin . Tryptic digestion of the fusion protein quantitatively produced big ET-1 (1-37), which is depleted in the C-terminal serine . However, a treatment of the fusion protein with 1,2-cyclohexanedione prior to tryptic digestion gave full-length big ET-1 with N7, -N8-(1,2-dihydroxycyclohex-1,2- ylene)-arginine . This modification was reversed to the intact arginine residue when the modified big ET-1 was incubated in 0.5 M TRIS-HCl buffer, pH 8.0 . Consequently, a combination of such a reversible protein modification and tryptic digestion gave 1.74 mg of recombinant big ET-1 from 2.01 of culture broth . The procedure described here may be applied to produce other arginine-containing peptides from fusion proteins.

FEMS Microbiol Lett, 1994 May 15, 118(3), 259 - 63
Expression of the Streptomyces griseus alpha-amylase gene in Escherichia coli; Vigal T et al.; The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E . coli RNA polymerase, as confirmed by using promoter-probe vectors . The expression of the amy gene in E . coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium . The extracellular alpha-amylase detected in the culture broth seems to be released by cellular lysis . When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no alpha-amylase activity was detected but larger E . coli cells and inclusion bodies were observed.

Appl Environ Microbiol, 1994 May, 60(5), 1630 - 4
Sodium chloride induces an NhaA/NhaR-independent acid sensitivity at neutral external pH in Escherichia coli; Rowbury RJ et al.; Escherichia coli previously grown in low-salt broth, pH 7.0, produced organisms which were markedly more acid sensitive when subsequently cultured in the same broth with 200 mM or more salt (NaCl) added . Induction of acid sensitivity occurred rapidly at both 37 and 30 degrees C, with a substantial effect within 15 min . Sensitization was partially inhibited by chloramphenicol and tetracycline and may depend on both protein synthesis-dependent and -independent physiological changes in the NaCl-induced organisms; sensitization did not result from osmotic shocking on transfer to challenge medium . Induction of acid sensitivity was affected by neither the sodium ion pore inhibitor amiloride nor the DNA synthesis inhibitor nalidixic acid; rifampin had a small effect, similar to that of chloramphenicol . Chlorides of other monovalent cations, especially Li+ and NH4+, also produced sensitization to acid, although CsCl was ineffective but did not interfere with sensitization by NaCl . Other sodium salts were also active as sensitizers, as were chlorides of divalent cations, but although sucrose (but not glycerol) was a good inducer, the results were not fully in accord with triggering of induction solely by the NaCl-associated increase in osmotic pressure . Sensitization was not prevented by deletion of the nhaA, nhaR, or nhaB gene . Acid sensitivity of NaCl-induced cells was slightly reduced after 90 min of growth at 37 degrees C in low-salt broth but was completely lost after 240 min . For NaCl-induced cells, acid killing in challenge media was not inhibited by amiloride . The NaCl-induced sensitization is distinct from the phenomenon of acid sensitivity induction in E . coli at alkaline external pH.

J Appl Bacteriol, 1994 Apr, 76(4), 412 - 6
The effect of electron beam irradiation and modified pH on the survival and recovery of Escherichia coli; Fielding LM et al.; The severity of radiation processing can be reduced by combining irradiation with other treatments, such as low pH . An exponential phase culture of Escherichia coli was irradiated at doses of 0-2.4 kGy at pH values ranging between 7.0 and 4.0, in an enriched nutrient broth . At pH 4.3 and above there was no significant effect of lowering the pH prior to irradiation . At pH 4.13 and 4.0, a much higher level of cell death occurred compared with irradiation at pH 7.0 . This synergistic effect was observed only when the pH was lowered before radiation processing.

J Bacteriol, 1994 Apr, 176(8), 2143 - 50
Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids; Pruss BM et al.; We isolated and characterized mutants defective in nuo, encoding NADH dehydrogenase I, the multisubunit complex homologous to eucaryotic mitochondrial complex I . By Southern hybridization and/or sequence analysis, we characterized three distinct mutations: a polar insertion designated nuoG::Tn10-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated delta(nuoFGHIJKL)-1 . Cells carrying any of these three mutations exhibited identical phenotypes . Each mutant exhibited reduced NADH oxidase activity, grew poorly on minimal salts medium containing acetate as the sole carbon source, and failed to produce the inner, L-aspartate chemotactic band on tryptone swarm plates . During exponential growth in tryptone broth, nuo mutants grew as rapidly as wild-type cells and excreted similar amounts of acetate into the medium . As they began the transition to stationary phase, in contrast to wild-type cells, the mutant cells abruptly slowed their growth and continued to excrete acetate . The growth defect was entirely suppressed by L-serine or D-pyruvate, partially suppressed by alpha-ketoglutarate or acetate, and not suppressed by L-aspartate or L-glutamate . We extended these studies, analyzing the sequential consumption of amino acids by both wild-type and nuo mutant cells growing in tryptone broth . During the lag and exponential phases, both wild-type and mutant cells consumed, in order, L-serine and L-aspartate . As they began the transition to stationary phase, both cell types consumed L-tryptophan . Whereas wild-type cells then consumed L-glutamate, glycine, L-threonine, and L-alanine, mutant cells utilized these amino acids poorly . We propose that cells defective for NADH dehydrogenase I exhibit all these phenotypes, because large NADH/NAD+ ratios inhibit certain tricarboxylic acid cycle enzymes, e.g., citrate synthase and malate dehydrogenase.

Mol Biol Evol, 1994 Mar, 11(2), 159 - 68
On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli; Hall BG; Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge . Examples of selection-induced mutations have been reported both in bacteria and in yeast . I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed . Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells . Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell . I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.

Mol Gen Genet, 1994 Mar, 242(5), 623 - 30
Analysis of the fimB promoter region involved in type 1 pilus phase variation in Escherichia coli; Schwan WR et al.; Many strains of Escherichia coli express type 1 pili which undergo phase variation during growth in broth, but become uniformly nonpiliated when passaged on agar media . In a previous analysis of these agar phase locked strains, we demonstrated that fimB and fimE, which mediate the site-specific DNA rearrangement involved in phase variation, are differentially transcribed under the two growth conditions . In this study the fimB promoter region was sequenced and characterized in agar phase locked strain J96 . Primer extension analysis of the fimB gene identified three putative transcription initiation sites . Transcription starting from two of the sites (P1 and P2) would produce an mRNA that approximates the transcript size of fimB previously detected by Northern blot hybridizations . Gel mobility shift studies revealed the presence of promoter binding activity in broth-grown cell extracts, but not in extracts from agar-grown cells, that reacted specifically with DNA fragments upstream of the P1 and P2 promoter regions . This putative binding protein may be involved in the regulation of fimB, perhaps by acting as a transcriptional activator.

J Bacteriol, 1994 Mar, 176(5), 1521 - 3
Escherichia coli RNA polymerase mutants that enhance or diminish the SOS response constitutively expressed in the absence of RNase HI activity; Kogoma T; Escherichia coli rnhA mutants lacking RNase HI chronically express the SOS response (T . Kogoma, X . Hong, G . W . Cadwell, K . G . Barnard, and T . Asai, Biochimie 75:89-99, 1993) . Seventeen rpoB (Rifr) mutant alleles, which encode altered beta subunits of RNA polymerase, giving rise to resistance to rifampin, were screened for the ability to enhance or diminish constitutive expression of the SOS response in rnhA mutants . Two mutations, rpoB3595 and rpoB2, were found to enhance the SOS response 5- and 2.5-fold, respectively, only when RNase HI is absent . These mutations rendered rnhA mutant cells very sensitive to broth; i.e., the plating efficiency of the double mutants was drastically reduced when tested on broth plates . Two mutations, rpoB8 and rpoB3406, were found to diminish constitutive SOS expression in rnhA mutants by 43 and 30%, respectively . It was suggested that RNA polymerase may have a property that influences the size of DNA-RNA hybrids, the frequency of their formation, or both and that the property resides at least in part in the beta subunit of the polymerase.

J Antibiot (Tokyo), 1994 Jan, 47(1), 37 - 45
Cyclothialidine, a novel DNA gyrase inhibitor . II . Isolation, characterization and structure elucidation; Kamiyama T et al.; Cyclothialidine is a novel DNA gyrase inhibitor produced by Streptomyces filipinensis NR 0484 . It was isolated from the culture broth by charcoal adsorption, Diaion HP-21, Amberlite CG-50, DEAE Toyopearl, and Toyopearl HW-40 SF column chromatography . The structure of cyclothialidine was determined to be a unique twelve membered lactone by amino acid analysis and various 2D-NMR experiments . Cyclothialidine inhibited Escherichia coli DNA gyrase with an IC50 of 30 ng/ml.

Appl Environ Microbiol, 1993 Dec, 59(12), 4347 - 9
Rapid glutamate decarboxylase assay for detection of Escherichia coli; Rice EW et al.; A rapid test procedure for the enzyme glutamate decarboxylase was developed for detection of Escherichia coli . The assay procedure was able to confirm the presence of E . coli in enteric broth cultures with 95% specificity for both pure cultures and environmental samples . The procedure was capable of detecting survivors among chlorine-exposed cells.

J Bacteriol, 1993 Nov, 175(22), 7160 - 9
Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein?
Ernsting BR, Denninger JW, Blumenthal RM, Matthews RG.
The regulon controlled by the leucine-responsive regulatory protein (Lrp) of Escherichia coli consists of over 40 genes and proteins whose expression is regulated, either positively or negatively, by Lrp . The gltBDF operon, encoding glutamate synthase, was originally identified as a member of the Lrp regulon through a two-dimensional electrophoretic analysis of polypeptides from isogenic strains containing or lacking a functional Lrp protein . We have now demonstrated that Lrp regulates the transcription of gltBDF::lacZ operon fusions . Relative to expression in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium, gltBDF::lacZ expression in an lrp+ strain is repressed 2.2-fold in the presence of 10 mM exogenous leucine and 16-fold in Luria broth . Repression of gltBDF::lacZ expression by leucine or Luria broth is not seen for an isogenic strain containing a Tn10 insertion in lrp, and expression of gltBDF::lacZ is 44-fold lower than in the lrp+ strain when both are grown in glucose minimal MOPS medium . Lrp binds specifically to DNA fragments containing the gltBDF promoter region . Saturating levels of leucine do not abolish binding of Lrp upstream of gltBDF but merely increase its apparent dissociation constant from 2.0 to 6.9 nM . Electrophoretic analysis of the Lrp regulon established that target proteins differ greatly in the degree to which the effect of Lrp on their expression is antagonized by leucine . On the basis of our present results, we present a model for positive regulation of target genes by Lrp . Insensitivity to leucine would be expected when the effective intracellular concentration of Lrp is high relative to the affinity of Lrp binding sites required for transcription of the target gene . At lower concentrations of Lrp, transcription of the target gene should be sensitive to leucine . This model suggests that regulation of the concentration of active Lrp is critical to control of the Lrp regulon.

Appl Microbiol Biotechnol, 1993 Nov, 40(2-3), 365 - 9
Purification of fusion proteins using affinity microspheres in aqueous two-phase systems; Kondo A et al.; Affinity microspheres were prepared by immobilizing human gamma-globulin (H gamma Gb) onto carboxylated poly (styrene/acrylamide) latex particles {P(St/AAm)-H; average diameter 0.33 microns}, which were prepared by emulsifier-free emulsion polymerization . H gamma GB was covalently immobilized onto the latex particles with high efficiency by the carbodiimide method . A fusion protein (ZZB1B2) of immunoglobulin G and albumin-binding domains (ZZ and B1B2, respectively) was expressed intracellularly and extracellularly in Escherichia coli and was purified by the affinity microspheres . In poly (ethylene glycol) (PEG)/potassium phosphate aqueous two-phase system, the affinity microspheres were partitioned into the PEG-rich top phase, while cells and cell debris of E . coli were displaced into the salt-rich bottom phase . Therefore, ZZB1B2 was directly purified from cell disintegrate or culture broth by combining the affinity microspheres with the aqueous two-phase partitioning, and its purity was almost the same as that purified by conventional affinity chromatography . Therefore, by this purification method, the primary purification process and the subsequent high resolution purification process are combined, and the number of purification steps can be reduced.

Mutat Res, 1993 Aug, 288(2), 311 - 9
The high mutator activity of the dnaQ49 allele of Escherichia coli is medium-dependent and results from both defective 3'-->5' proofreading and methyl-directed mismatch repair; Krishnaswamy S et al.; The Escherichia coli dnaQ49 mutator allele maps at the dnaQ locus, the structural gene for the epsilon subunit of the DNA polymerase III holoenzyme . Epsilon, when bound to the alpha subunit, provides the 3'-->5' exonuclease activity (proofreading) that removes 3' mismatched terminal nucleotides from the nascent DNA strand during replication . The temperature sensitive dnaQ49 allele lacks this catalytic activity which results in mutation frequencies 10(4)-10(5)-fold above wild-type values at 37 degrees C . At 30 degrees C dnaQ49 mutation frequencies are much lower but still higher than wild-type levels . We found that dnaQ49, like mutD5, another strong mutator allele of dnaQ, is medium-dependent with mutation frequencies ranging from 12 to nearly 1000-fold higher in rich media (L-broth) than in minimal media . In minimal media dnaQ49 retains modest mutator activity . In addition the base-pair substitution mutational spectrum of dnaQ49 was medium-dependent . Unlike mutD5 the addition of thymidine to minimal medium did not enhance dnaQ49 mutator activity . We also constructed dnaQ49mutL double mutator strains and compared mutator frequencies with single dnaQ49 and mutL strains . The mutL allele results in inactive methyl-directed mismatch repair . Double and single dnaQ49 mutators had similar mutation frequencies at 37 degrees C in L-broth suggesting that dnaQ49 strains are defective in mismatch repair as well as 3'-->5' exonuclease proofreading activity . In contrast in minimal media at 37 degrees C and in L-broth at 30 degrees C dnaQ49 mutL mutation frequencies were much higher than dnaQ49 values indicating the presence of active mismatch-repair activity in the latter strain . In addition at 37 degrees C dnaQ49mutL mutation frequencies were about 100-fold higher in L-broth than in minimal media . We conclude from this result that the rich media effect with dnaQ49 involves an actual increase in replication errors rather than a medium-dependent modulation of mismatch repair activity.

Avian Dis, 1993 Jul-Sep, 37(3), 763 - 6
Effect of spectinomycin on Escherichia coli infection in 1-day-old ducklings; Freed M et al.; Two challenge trials and one confirmation trial were conducted to evaluate the efficacy of spectinomycin in the treatment of 1-day-old ducklings infected with Escherichia coli . In the challenge trials, ducklings were injected in the right posterior thoracic air sac with 0.2 cm3 of broth containing 10(8) colony-forming units E . coli (strain O78, E38)/ml . Spectinomycin at dosage levels of 2.5 mg, 5.0 mg, and 10.0 mg of activity was injected subcutaneously 6 hours following infection . The confirmation trial was conducted to confirm the challenge trials; procedures were similar to those used in the challenge trials, except that only the 5.0 mg of activity dosage of spectinomycin was used . In both types of trials, spectinomycin-treated ducklings had significantly lower mortality and higher average weight gain, average daily gain, and feed consumption than infected unmedicated controls . These results indicate that spectinomycin is effective in treating ducks for experimentally induced colibacillosis caused by E . coli (strain O78, E38).

Infect Immun, 1993 Jun, 61(6), 2563 - 9
Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli; Hu LT et al.; The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli . Enzymatic activity, however, has been very weak compared with that in clinical isolates of H . pylori . Conditions under which near wild-type urease activity was achieved were developed . E . coli . SE5000 containing recombinant H . pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808) . Under these conditions, E . coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H . pylori UMAB41 (100 U/mg of protein), from which the genes were cloned . Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine . In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein . As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids . We conclude that recombinant H . pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided.

J Med Microbiol, 1993 Apr, 38(4), 256 - 61
The induction of trimethoprim resistance encoded by the type IV dihydrofolate reductase gene; Young HK et al.; The effect of plasmid pUK1123, which confers low level resistance to trimethoprim when tested on solid minimal medium, but also no resistance when tested on IsoSensitest agar, was investigated in liquid media . The growth of Escherichia coli J62-2, harbouring pUK1123, was unaffected in liquid minimal medium containing trimethoprim 10 mg/L . However, in IsoSensitest broth, exposure to this drug concentration resulted in bacteriostasis . After an initial delay, resistance to trimethoprim was induced in IsoSensitest broth containing trimethoprim 10 mg/L, by the imposition of thymine starvation . This response was immediately reversible when trimethoprim was removed, confirming that resistance resulted from induction rather than selection of resistant mutants.

J Bacteriol, 1993 Apr, 175(8), 2236 - 40
Mechanism of adverse conditions causing lack of flagella in Escherichia coli; Shi W et al.; Escherichia coli lacks flagella when grown in tryptone broth in the presence of various adverse conditions (C . Li, C . J . Louise, W . Shi, and J . Adler, J . Bacteriol . 175:2229-2235, 1993) . Now, the synthesis, rather than the degradation, of flagellin was shown to be inhibited . Studies of transcriptional fusions of flagellar operons to the lacZ gene revealed that transcription of the flagellar genes was reduced in cells grown under these adverse conditions . Increasing gene dosage of the flhD operon by a plasmid partially suppressed the nonflagellation caused by some adverse conditions . The signal which shuts off the synthesis of flagella under adverse conditions remains to be discovered . This shutting-off process does not result from catabolite repression or from signals from the chemotaxis system.

Appl Microbiol Biotechnol, 1993 Apr, 39(1), 48 - 52
Cultivation of Escherichia coli to high cell densities in a dialysis reactor; Markl H et al.; High cell density cultivation of Escherichia coli on a glycerol-based mineral medium was studied . The cultivation was done in a dialysis reactor composed of two chambers . The inner chamber is formed and separated from an outer chamber by a membrane . Fresh medium was continuously exchanged with medium in the outer chamber so that both glycerol and other components of the medium were supplied to the inner chamber through the membrane . Inhibitory substances diffused from the inner to the outer chamber and were subsequently removed with effluent from the outer chamber . Initially, mathematical models were used to describe the process . The optimal cultivation parameters, such as the initial glycerol concentrations in the two chambers, the desired transport rate across the membrane, glycerol concentration in the feed/dialysing medium, and the time to start the medium exchange, were determined from preliminary experiments and calculations . The actual cultivation results agreed very well with the model predictions . A very high cell concentration of 174 g dry weight/l was obtained . This cell concentration is within the range of the maximum theoretical concentration of E . coli in culture broth (160-200 g/l).

Appl Environ Microbiol, 1993 Mar, 59(3), 663 - 8
In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin; Bradburne JA et al.; The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo . Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions . Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins . NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ) . Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase) . The labeling procedure was also used to label E . coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose . Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations . In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose . OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E . coli cells grown in medium containing 20% sucrose . Coomassie-stained gels, however, revealed similar quantities of OmpF in E . coli cells grown at all three sucrose concentrations . These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.

Infect Immun, 1993 Mar, 61(3), 1126 - 31
Aggregative adherence fimbria I expression in enteroaggregative Escherichia coli requires two unlinked plasmid regions; Nataro JP et al.; Adherence to HEp-2 cells by many enteroaggregative Escherichia coli (EAggEC) strains is associated with the expression of flexible, bundle-forming fimbriae 2 to 3 nm in diameter, designated aggregative adherence fimbriae I (AAF/I) . We have previously reported the molecular cloning and TnphoA mutagenesis of AAF/I genes from the large plasmid of prototype EAggEC strain 17-2 (J . P . Nataro, Y . Deng, D . R . Maneval, A . L . German, W . C . Martin, and M . M . Levine, Infect . Immun . 60:2297-2304, 1992) . Here, we report that further mapping and subcloning of AAF/I regions suggest that expression of the fimbriae requires two separate plasmid regions (designated regions 1 and 2) . Approximately 9 kb of DNA unnecessary for fimbrial expression separates the two regions; this intervening segment encodes the EAggEC heat-stable enterotoxin (EAST1) . Neither region was capable of conferring aggregative HEp-2 adherence (AA) when cloned individually; when the two regions were cloned as a single fragment or when each was cloned into a different vector and introduced into the same E . coli HB101 cell, AA was restored . AA-positive constructs also expressed human erythrocyte hemagglutination, autoagglutination in broth cultures, and the production of AAF/I as detected by immunogold electron microscopy.

Environ Mol Mutagen, 1993, 22(3), 157 - 63
Modulation of the H2O2-induced SOS response in Escherichia coli PQ300 by amino acids, metal chelators, antioxidants, and scavengers of reactive oxygen species; Muller J et al.; The SOS chromotest is a simple colorimetric genotoxicity assay that monitors DNA repair by measuring the induction of the gene sfiA in Escherichia coli K-12 . E . coli PQ300, a diagnostic SOS tester strain for the detection of oxidative genotoxins, carries a mutation in a key gene for antioxidative defense, oxyR . This mutation renders PQ300 more sensitive to oxidative genotoxins, particularly to H2O2 . We found that induction of the SOS response by H2O2 in E . coli PQ300 is dependent on the composition of the incubation medium; a substantially reduced response was obtained in minimal phosphate buffered saline (PBS) as opposed to complex Luria broth (LB) medium . Supplementation of PBS with histidine or cysteine stimulated H2O2-induced SOS induction to levels exceeding those found in LB medium . Low concentrations of glutathione (20-70 microM) also enhanced the H2O2-induced SOS response in E . coli PQ300, whereas higher concentrations (> 150 microM) were protective . Preincubation of tester cells with the chelators o-phenanthroline, 2,2-dipyridyl, and ethylenediaminetetraacetic acid (EDTA) protected cells from the effects of H2O2, although EDTA was only partially effective . Pretreatment of PQ300 with the antioxidant ascorbic acid or the hydroxyl radical scavenger dimethyl sulfoxide also diminished the SOS response, whereas mannitol and glucose were ineffective . The results show that the net effect of H2O2-induced DNA damage is influenced by the balance of oxidative and antioxidative factors and, furthermore, can be modulated by constituents of the extracellular milieu.

Biochimie, 1993, 75(1-2), 89 - 99
Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities; Kogoma T et al.; rnhA224 and rnhA339::cat mutants which lack RNase HI activity were found to constitutively express the sfiA::lacZ operon fusion in a recA+ lexA(+)-dependent manner . The sfiA::lacZ expression (indicating SOS induction) in rnhA mutants was increased to higher levels by the introduction of the recD1903 or recB21 mutation . The SOS induction in these cells was further enhanced by nutritional shift up from casamino acid medium to Luria broth . Although the extent by which the recD and recB mutations increased the sfiA expression in rnhA mutants was similar, the rnhA224 recB21 double mutant had plating efficiencies that were 25-fold lower on casamino acid plates and 5 x 10(5)-fold lower on Luria broth plates than the respective plating efficiencies of either rnhA224 recD or rnhA::cat recD double mutants . Whereas the recD mutation inactivates the exonuclease activity of the RecBCD (Exo V) enzyme without reducing the recombination proficiency of the mutant, the recB21 mutation abolishes both the exonuclease activity and recombination capability . Therefore, in the absence of both RNase HI and Exo V activities, homologous recombination functions become crucial for viability, particularly in Luria broth . Introduction of mutations in recA, recJ and recN exacerbated the phenotypes . It is proposed that R-loops which persist due to the lack of RNase HI activity can be removed by two alternative routes of DNA repair: one involving Exo V, Exo I and DNA polymerase I, and the other involving both the RecBCD and RecF pathways of homologous recombination activities . The isolation of RNA polymerase mutants that constitutively express the SOS response at high levels and exhibit remarkable broth-sensitivity lend strong support to the contention that increased amounts of the persisting R-loop in rnhA mutants growing in Luria broth give rise to a stronger SOS response.

Microbios, 1993, 76(306), 47 - 54
Comparison of fluorogenic and conventional membrane filter media for enumerating coliform bacteria; Cenci G et al.; 4-Methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide were added to MacConkey broth and their diagnostic powers for total coliforms (TC) and Escherichia coli, respectively, were tested by membrane filtration at primary isolation . Examining water samples from different sources proved the usefulness of fluorogenic rather than reference media both as regards recovery efficiency and rapidity (possible within 12 h) of analyses . The recoveries obtained by fluorogenic and conventional tests for both TC and E . coli were correlated . Values were comparable in surface water samples, while a higher sensitivity of fluorogenic media was observed in samples of shallow contaminated ground water . Results seem to indicate that the use of fluorogenic membrane filtration analysis for colimetric indicators could be favourably considered especially for sanitary surveying of drinking water.

IEEE Trans Biomed Eng, 1992 Dec, 39(12), 1310 - 3
Impedance bacteriometry: medium and interface contributions during bacterial growth; Felice CJ et al.; We measured impedance in a cell containing culture broth inoculated with E . coli, before and during bacterial growth . The electrode interface impedance components (Ri, Xi) and the culture medium component Rm were separated by making use of the Warburg's model frequency dependent properties . Measurements were carried out at different frequencies (from 18 Hz to 18 kHz) with a constant current impedance bridge as growth proceeded . It was found that: Growth curves for Ri and Xi showed a similar temporal pattern within the frequency range of 18-100 Hz . Dispersion was not observed in Rm, meaning that the same growth response was obtained within the 18-18,000 Hz range . At low frequency, the resistive and capacitive reactive components, or Rb and Xb, respectively, were directly measured, where Rb = (2.Ri + Rm) and Xb = 2.Xi and, at high frequency (above 5 kHz), Rm was obtained (for Zi is negligible) . Thus, Ri was easily discriminated from Rm by simple arithmetic: Ri = {Rb (low f) - Rb (high f)}/2 . In four experiments, the maximum spread of Xi, Ri, and Rm was smaller than 5%, indicating good repeatability . There is potential new information in dissecting out the growth curve in three separate component curves.

J Biochem (Tokyo), 1992 Sep, 112(3), 360 - 5
High-yield production of recombinant endothelin-1; Yasufuku K et al.; A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography . Trypsin digestion of the purified pETB-42P gave big ET-1(1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1(1-37) gave ET-1(1-21) in a yield of 74% (overall yield: 52%) . Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60% . One milligram of ET-1 or 2.0 mg of big ET-1(1-37) was obtained from 1.8 liters of culture broth . Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1(1-37) had almost the same in vitro and in vivo biological activities as big ET-1(1-38).

Appl Environ Microbiol, 1992 Aug, 58(8), 2704 - 7
Temperature-dependent induction of an acid-inducible stimulon of Escherichia coli in broth; Hassani M et al.; The induction of the inducible lysyl-tRNA synthetase, LysU, and the inducible lysine and arginine decarboxylases of Escherichia coli K-12 grown in AC broth to a pH of 5.5 or less is temperature dependent, being distinctly lower at 24 than at 37 degrees C . This induction does not appear to be under HtpR control.

J Bacteriol, 1992 Aug, 174(16), 5190 - 5
Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids; Austin SJ et al.; The maintenance of partition-defective (Par-) mini-P1 and mini-F plasmids was studied in topA strains of Escherichia coli, which are defective in topoisomerase I activity . The partition defects were substantially but not completely suppressed in broth-grown cultures . This suppression was not due to a large increase in copy number . However, the absolute number of copies of Par- mini-P1 plasmids per average dividing cell is sufficiently high to account for the modest stability observed if a random distribution of the copies to daughter cells is assumed . The similar number of Par- plasmid copies in wild-type cells are distributed in a considerably worse-than-random fashion . Thus, it is unnecessary to propose, as was suggested previously, that an active, par-independent pathway operates in topA strains to ensure proper segregation of the plasmids to daughter cells . Rather, it seems likely that the lack of topoisomerase I activity aids the random distribution of the partition-defective plasmids, perhaps by facilitating their separation after replication . The results of studies carried out at reduced growth rates were consistent with this view; when topA cells containing Par- mini-P1 plasmids were cultured in minimal medium, in which the copy number of the plasmids per average cell is sharply reduced, very little suppression of the partition defect was observed.

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