Plasmid-mediated gentamicin resistance of Pseudomonas aeruginosa and its lack of expression in Escherichia coli

We isolated 11 nonconjugative plasmids mediating resistance to aminoglycoside antibiotics, including gentamicin, from Pseudomonas aeruginosa strains. Their genetic properties were investigated in both P. aeruginosa and Escherichia coli transformants. The plasmid molecular weights ranged from 11 x 10(6) to 24 x 10(6). A low level or complete absence of gentamicin resistance was observed when these plasmids were introduced into E. coli, but gentamicin resistance was restored when the plasmids were transferred back to P. aeruginosa from E. coli. Aminoglycoside-modifying enzyme activity was detected in P. aeruginosa harboring these plasmids, but was absent or greatly reduced in E. coli strains. This lack of expression may explain the observed decrease in aminoglycoside resistance.     

A glutamate-dependent acid resistance gene in Escherichia coli

Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 isolates screened, 3 Tet(r) strains were identified as extremely acid sensitive (<0.1% survival at pH 2.5 for 2 h). Flanking sequences of the Tn10 inserts were amplified by inverse PCR. The sequences encoded a hydrophobic partial peptide of 88 residues. A random-primer-generated probe hybridized to Kohara clones 279 and 280 at 32 min (33.7 min on the revised genomic map EcoMap7) near gadB (encoding glutamate decarboxylase). The gene was designated xasA for extreme acid sensitive. xasA::Tn10 strains grown at pH 7 to 8 showed 100-fold-less survival in acid than the parent strain. Growth in mild acid (pH 5 to 6) restored acid resistance; anaerobiosis was not required, as it is for acid resistance in rpoS strains. xasA::Tn10 eliminated enhancement of acid resistance by glutamic acid. xasA was found to be a homolog of gadC recently sequenced in Shigella flexneri, in which it appears to encode a permease for the decarboxylated product of GadB. These results suggest that GadC (XasA) participates in a glutamate decarboxylase alkalinization cycle to protect E. coli from cytoplasmic acidification. The role of the glutamate cycle is particularly important for cultures grown at neutral pH before exposure to extreme acid.     

Detection and characterization of the fimA gene of Escherichia coli O157:H7

An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E. coli strains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared by E. coli O157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of the fimA region demonstrated that E. coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E. coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E. coli O157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E. coli O157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detect E. coli strains of the O157:H7 serotype.     

Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization

OBJECTIVES: To define the incidence and severity of ovarian hyperstimulation syndrome (OHSS) occurring in oocyte donors. METHODS: Women (n = 149) aged 31.3 +/- 4.8 years (mean +/- S.D., range 21-41 years) participated as designated oocyte donors and underwent 400 consecutive cycles of controlled ovarian stimulation using human menopausal gonadotropin following pituitary downregulation with gonadotropin-releasing agonist. Patients were monitored by serial transvaginal ultrasound examinations and serum estradiol (E2) determinations. Oocytes (15.6 +/- 7.5 per aspiration; range 2-57) were harvested by ultrasound-directed transvaginal follicle aspiration 36 h following the intramuscular injection of human chorionic gonadotropin (hCG). Follow-up examination occurred 1 and 2 weeks post-aspiration. RESULTS: On the day of hCG injection E2 levels ranged from 512 to 13,502 pg/ml (mean 2902.7 +/- 1486.9 pg/ml). Over the next few weeks the degree of hyperstimulation in donors was staged: mild 65% (grade I, n = 98; grade II, n = 162); moderate 33.5% (grade III, n = 120; grade IV, n = 14); severe 1.5% (grade V, n = 6; grade VI, n = 0). Associated preaspiration E2 levels were: grade I, 1120 +/- 424 pg/ml; grade II, 2084 +/- 613 pg/ml; grade III, 3785 +/- 1713 pg/ml; grade IV, 5370 +/- 1264 pg/ml; grade V, 4286 +/- 1100 pg/ml. Worsening OHSS was associated with increasing levels of E2. There were no serious complications and hospitalization was not required. All symptoms resolved within 30 days of aspiration, disappearing by the time of the first menstrual flow in women of grade-III or lower stage. CONCLUSION: Although oocyte donors commonly experienced exaggerated levels of serum E2 they rarely (< 2%) developed severe OHSS. This may be attributable to their lack of embryo transfer which avoids exacerbating the illness.     

Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase

The present studies were designed to evaluate the developmental toxicity of chlorpropham in mice. The first study was conducted to determine administration time, and the second study was designed to evaluate dose-response effects. Chlorpropham was administered to pregnant mice by gavage on Days 8, 8.3, 9, 9.3, 10, and 11 of gestation at a level of 3000 mg/kg bw, and the females were killed on Day 18 of gestation. The administration on Day 8.3 of gestation induced the highest percentage of external malformations with brachyury occurring among more litters than in other groups. Chlorpropham was administered to pregnant mice by gavage at a level of 0 (control), 750, 1500, and 3000 mg/kg bw on Day 8.3 of gestation, and the females were killed on Day 18 of gestation. The total resorption rate was significantly increased in the 3000 mg/kg bw group. The average fetal body weight of each sex was significantly reduced in the 3000 mg/kg treatment group. The total incidence of external malformations was significantly increased in the two highest dose groups in a dose-related manner. Again brachyury was significantly increased in the 3000 mg/kg bw group.     

Purification and characterization of D-beta-hydroxybutyrate dehydrogenase expressed in Escherichia coli

D-beta-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 mumol/(min.mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.     

Isolation and characterization of the heat-responsive genes in Escherichia coli

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKm vs pH were different for the wt and H356K variant. Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.     

uspB, a new sigmaS-regulated gene in Escherichia coli which is required for stationary-phase resistance to ethanol

The open reading frame immediately upstream of uspA is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions. UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains. Overexpression of UspB causes cell death in stationary phase, whereas mutants of uspB are sensitive to exposure to ethanol but not heat in stationary phase. In contrast to uspA, stationary-phase induction of uspB requires the sigma factor sigmaS. The expression of uspB is modulated by H-NS, consistent with the role of H-NS in altering sigmaS levels. Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon's previously known role in thermotolerance, osmotolerance, and oxidative stress resistance.     

Expression of the Streptomyces griseus alpha-amylase gene in Escherichia coli

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.     

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.     

Protein translocation functions of Escherichia coli SecY: in vitro characterization of cold-sensitive secY mutants

Activated macrophages are among the major constituents of the periapical granuloma. Their state of activation may persist for long periods after the local irritant is removed and may delay resolution and repair of the lesion. The effect of activated macrophages on fibroblast growth was studied in vitro. Circular fibroblast colonies were formed using a drop containing 7.5 x 10(5) murine dermal fibroblasts and allowed to grow for 7 days. When peritoneal exudate macrophages were added (0.5-3.0 x 10(6) cells/dish) and activated in vitro by LPS (1 microgram/ml), the fibroblast colony's growth was suppressed. LPS alone, at the concentration used, had no effect on the fibroblast growth. Hydrocortisone (> or = 10(-7) M) totally reversed the suppression, when added either simultaneously with or 6, 24, or 48 h after the LPS. The efficacy of late hydrocortisone treatment suggests that its effect was through prevention of the expression of the LPS activation of the macrophages. These findings may provide a possible clue to a pharmacological modulation of the healing processes that occur in the periapical lesion once its infective source had been eliminated.     

Purification, characterization, and crystallization of Escherichia coli ribokinase

Ribokinase phosphorylates ribose to form ribose-5-phosphate in the presence of ATP and magnesium. The phosphorylated sugar can enter the pentose phosphate pathway or be used for the synthesis of nucleotides, histidine, and tryptophan. Ribokinase belongs to the PfkB family of carbohydrate kinases, for which no three-dimensional structure is currently known. We describe an improved purification protocol for Escherichia coli ribokinase and give evidence from light-scattering and gel filtration studies that the protein forms a dimer in solution. Several types of crystals are also described that have been obtained of apo ribokinase, ribokinase in the presence of ATP, and in a ternary complex with an ATP-analogue and ribose. The latter crystals give the best X-ray diffraction. A complete data set has been collected at the synchrotron source in Hamburg, to 2.6 A resolution using a frozen crystal. The crystals belong to space group P6(1)22 or P6(5)22 with cell parameters a = b = 95 A and c = 155 A.     

Computer identification of Escherichia coli rRNA gene restriction patterns

Hydropic vacuolation (HydVac) of biliary epithelial cells and hepatocytes is described for 3 species of U.S. West Coast bottom fishes. Risk assessment analyses are also conducted to determine if the prevalence of this lesion increases in association with contaminant exposure and site of capture. The morphology of HydVac in starry flounder Platichthys stellatus, white croaker Genyonemus lineatus and rock sole Lepidopsetta bilineata was similar to that described in winter flounder Pleuronectes americanus from the U.S. Atlantic Coast, especially in that HydVac most commonly affected biliary epithelial cells. Hydropic vacuolation was the most prevalent liver lesion in starry flounder and white croaker captured from contaminated environments. Risk assessment analyses confirmed that the relative risk for HydVac increased with the presence of aromatic and chlorinated hydrocarbons in sediment, fish bile, and fish liver for these species. Hydropic vacuolation also frequently occurred in rock sole, but the lesion showed no clear association with contaminant exposure in this species. The types of liver lesions that were useful biomarkers of contaminant effects in fish depended on the species and this factor must be taken into account when evaluating histopathological biomarkers of response to contaminant exposure.     

Construction and characterization of avian Escherichia coli cya crp mutants

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.     

Recombinant human phenylethanolamine N-methyltransferase: overproduction in Escherichia coli, purification, and characterization

We studied major malformations in 5,581 infants with Down syndrome (DS) from three registers of congenital malformations. THe prevalence at birth of 23 different malformations was compared with the program-specific rates for each malformation in non-DS infants. An about 300 times risk increase was seen for annular pancreas, cataracts and duodenal atresia and an about 100 times risk increase for megacolon and small choanal atresia. Esophageal, anal and small bowel atresia, preaxial polydactyly, and omphalocele all showed risk increases between 10 and 30 times. Statistically significantly elevated risk ratios around 3-5 were seen for cleft palate, cleft lip/palate, and limb deficiencies. No increased risk was seen for neural tube defects, hydrocephaly, microtia, renal agenesis or severe dysgenesis, hypospadias or polydactyly other than preaxial. Oral clefts were more often present in DS in the Swedish material than in the other two materials. Cardiac defects were registered in 26% of all cases (varying between programs) but 28% of the cardiac defects were unspecified. DS infants born to women younger than 25 years had a significantly increased risk for megacolon and there was a trend increasing risk for esophageal or anal atresia with maternal age. A decreased risk for cardiac defect in DS infants born to teenage mothers was found, quite pronounced for endocardial cushion defects and ventricular septum defects. There were no statistically significant differences in the sex distribution of specific malformations in infants with DS and in non-DS infants.     

Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli

We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells.     

Ten-year survey of quinolone resistance in Escherichia coli causing urinary tract infections

Two patients with central nervous system manifestations of Langerhans cell histiocytosis, both with brain stem involvement, are reported. The onset of symptoms was at an age when the diagnosis might not have been considered.