Showing papers on "Escherichia coli published in 1992"
TL;DR: The recent elucidation of the three-dimensional structure of the heat-labile enterotoxin has provided an opportunity to examine and compare the correlations between structure and function of the two toxins, which may improve understanding of the disease process itself and illuminate the role of the toxin in studies of signal transduction and G-protein function.
863 citations
TL;DR: The existence of similar proteins, heretofore with no known function, in bacterial species distantly related to Escherichia coli suggests that Dps may define a novel class of widely conserved DNA-binding proteins.
Abstract: A starvation-inducible DNA-binding protein was discovered as a result of the analysis of proteins synthesized in 3-day-old cultures of Escherichia coli. This 19-kD protein, designated Dps, is abundant in starved cells. In vitro, Dps forms extremely stable complexes with DNA, without apparent sequence specificity. When complexed with Dps, DNA is rendered DNase resistant. Mutant cells lacking Dps show dramatic changes in the pattern of proteins synthesized during starvation. The mutants also fail to develop starvation-induced resistance to hydrogen peroxide, an agent that can cause oxidative damage to DNA in vivo. These results have prompted us to postulate that Dps plays an important role both in gene expression and DNA protection during stationary phase. The existence of similar proteins, heretofore with no known function, in bacterial species distantly related to Escherichia coli suggests that Dps may define a novel class of widely conserved DNA-binding proteins.
767 citations
TL;DR: The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single-and double-stranded DNA as discussed by the authors.
Abstract: The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single- and double-stranded DNA. Two smaller subdomains at the N and C termini protrude from the protein and respectively stabilize a 6(1) helical polymer of protein subunits and interpolymer bundles. This polymer structure closely resembles that of recA/DNA filaments determined by electron microscopy. Mutations in recA protein that enhance coprotease, DNA-binding and/or strand-exchange activity can be explained if the interpolymer interactions in the crystal reflect a regulatory mechanism in vivo.
741 citations
TL;DR: An Escherichia coli chromosomal DNA fragment cloned on a multicopy plasmid conferred resistance to carbonylcyanide m-chlorophenylhydrazone, nalidixic acid, and a number of other toxic compounds.
Abstract: An Escherichia coli chromosomal DNA fragment cloned on a multicopy plasmid conferred resistance to carbonylcyanide m-chlorophenylhydrazone, nalidixic acid, and a number of other toxic compounds. The sequence of the cloned emr locus located at minute 57.5 of the chromosome revealed two open reading frames, emrA and emrB. emrB encodes a highly hydrophobic 56.2-kDa peptide, with 14 potential alpha-helices to span the inner membrane. The peptide is homologous to QacA, a multidrug-resistant pump from Staphylococcus aureus, and belongs to a gene family that includes tetracycline-resistant pumps of Gram-positive bacteria and the galactose/H+ symporter of E. coli. emrA encodes a putative 42.7-kDa peptide containing a single hydrophobic domain and a large C-terminal hydrophilic domain. An active pho-fusion to the C domain suggested that EmrA is a membrane protein. Disruption of emrB significantly increased sensitivity of cells to uncouplers. The cellular content of uncoupler increased in the order: overexpressed emrB cells greater than wild type greater than emrB-.
407 citations
TL;DR: It is demonstrated that protein F is an important adhesin of S. pyogenes, a bacterial surface protein that binds fibronectin at high affinity and had a much lower capacity to adhere to respiratory epithelial cells.
Abstract: Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.
351 citations
TL;DR: The PCR method, which is highly specific and easier to perform than the hybridization method, is a powerful genotypic assay for detection of adhesin-encoding operons and can be recommended for clinical use to detect virulent urinary E. coli strains, as well as for epidemiological studies.
Abstract: Adhesin-encoding operons (pap, sfa/foc, and afa) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. A quick and sensitive assay to identify these operons was developed by using the polymerase chain reaction (PCR). Three pairs of 25-mer primers were defined from the sequences of the DNA fragments used as probes in hybridization studies to identify each of the three operons, and the six primers were used together in a single reaction of amplification. To validate the PCR approach for detection of adhesin-encoding operons among clinical isolates, we investigated a collection of 97 E. coli isolates with the following characteristics: all isolates originated from the urine of patients with pyelonephritis, and the adhesin responsible for specific binding of the isolates to uroepithelial cells was previously characterized by phenotypic assays, as well as genotypic tests based on hybridization. There was a perfect correlation between the results obtained with the PCR approach and those previously obtained by using DNA probes. These results indicate that the PCR method, which is highly specific and easier to perform than the hybridization method, is a powerful genotypic assay for detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as for epidemiological studies.
333 citations
TL;DR: Using this reagent, the cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter and it is found that aberrant expression of p53 is a frequent feature of human breast cancer.
Abstract: A cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter. The cDNA was adapted so that the full-length protein was produced without fusion to any other sequence. Large amounts of the protein were isolated and the purified protein used to produce very high titre polyclonal antibodies to p53. These new antibodies permit the sensitive detection of p53 and p53 complexes in ELISA and immunoblotting assays. Most importantly, they also permit the detection of p53 in archival tumour material that has been conventionally fixed in formalin and embedded in paraffin wax. Using this reagent we have found that aberrant expression of p53 is a frequent feature of human breast cancer. We are able to recognise six different classes of p53 expression pattern that may be of help in the subclassification of breast tumours.
330 citations
TL;DR: The balance between inactive and active aconitase in growing, aerobic, Escherichia coli represented a pseudoequilibrium between inactivation by O2- and reactivation by restoration of Fe(II), and it provided a measure of the steady-state concentration of O1- within E. coli.
330 citations
TL;DR: While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity.
Abstract: A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.
319 citations
TL;DR: It is suggested that SP-D plays important roles in the lung's defense against Gram-negative bacteria.
Abstract: Surfactant protein D (SP-D) is a collagenous glycoprotein that is secreted into the pulmonary airspaces by alveolar type II and nonciliated bronchiolar cells. SP-D exhibits Ca(++)-dependent carbohydrate binding in vitro and is structurally related to the collagenous C-type lectins, including serum conglutinin, serum mannose-binding proteins, and surfactant protein A. Preliminary studies showed calcium- and saccharide-dependent binding of fluorescein-conjugated or radioiodinated SP-D to a variety of microorganisms, including Gram-negative bacteria and fungi. A laboratory strain of Escherichia coli (Y1088) was chosen to further examine the mechanism(s) of binding. Binding of SP-D to Y1088 was time dependent, saturable, and inhibited by cold SP-D or competing saccharides; Scatchard analysis gave a Kd of 2 x 10(-11) M. At higher concentrations, SP-D also caused Ca(++)-dependent agglutination of Y1088 that was inhibited by alpha-glucosyl-containing saccharides, antisera to the carbohydrate-binding domain of SP-D, or Y1088 LPS. Lectin blots showed specific binding of 125I-SP-D to Y1088 LPS, as well as LPS from other several strains of enteric Gram-negative bacteria. Immunogold studies demonstrated strong and uniform surface labeling of the bacteria. Rat and human bronchoalveolar lavage (BAL) caused Ca(++)-dependent agglutination of E. coli that was dose dependent and inhibited by competing saccharides or anti-SP-D. SP-D was selectively and efficiently adsorbed from rat BAL by incubation with E. coli, and incubation of E. coli with radiolabeled rat type II cell medium revealed that SP-D is the major E. coli-binding protein secreted by freshly isolated cells in culture. We suggest that SP-D plays important roles in the lung's defense against Gram-negative bacteria.
313 citations
TL;DR: It is proposed that E. coli messages beginning with a single-stranded RNA segment of significant length are preferentially targeted by a degradative ribonuclease that interacts with the mRNA 5' terminus before cleaving internally at one or more distal sites.
Abstract: The 5'-untranslated region of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer capable of prolonging the lifetime in E. coli of a number of heterologous messages to which it is fused. To elucidate the structural basis of differential mRNA stability in bacteria, the domains of the ompA 5'-untranslated region that allow it to protect mRNA from degradation have been identified by mutational analysis. The presence of a stem-loop no more than 2-4 nucleotides from the extreme 5' terminus of this RNA segment is crucial to its stabilizing influence, whereas the sequence of the stem-loop is relatively unimportant. The potential to form a hairpin very close to the 5' end is a feature common to a number of stable prokaryotic messages. Moreover, the lifetime of a normally labile message {bla mRNA) can be prolonged in £. coli by adding a simple hairpin structure at its 5' terminus. Accelerated degradation of ompA mRNA in the absence of a 5'-terminal stem-loop appears to start downstream of the 5' end. We propose that E. coli messages beginning with a single-stranded RNA segment of significant length are preferentially targeted by a degradative tibonuclease that interacts with the mRNA 5' terminus before cleaving internally at one or more distal sites.
TL;DR: The properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.
Abstract: 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies. Once 8-oxodGTP is formed in the cellular nucleotide pool, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein of Escherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified > 400-fold. Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a Km value of 12.5 microM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.
TL;DR: An EPEC plasmid‐encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence, is described.
Abstract: Summary
Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle-forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non-invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N-terminal amino acid sequence of the major structural protein of the bundle-forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle-forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phophatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid-encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.
TL;DR: Results demonstrate that BPI recognizes the highly conserved lipid A region of bacterial LPS via residues contained within the amino-terminal portion of the BPI molecule.
Abstract: Bactericidal/permeability-increasing protein (BPI) is a 55-kDa cationic protein (nBPI55) elaborated by polymorphonuclear neutrophils (PMN). BPI has potent bactericidal activity against a wide variety of gram-negative organisms and neutralizes endotoxin activities. An N-terminal fragment of nBPI55 exhibits the bactericidal and antiendotoxin properties of the holoprotein. To further characterize the biological activities of the N-terminal fragment, a recombinant protein (rBPI23) corresponding to the first 199 amino acids of human BPI was produced and purified. rBPI23 had antibacterial activity equivalent to that of nBPI55 against Escherichia coli J5. Furthermore, both rBPI23 and nBPI55 bound identically to a broad range of R- and S-form lipopolysaccharides (LPS) and to natural and synthetic lipid A. Binding of radiolabeled nBPI55 to LPS was inhibited in an identical fashion by either nBPI55 or rBPI23. The binding of both proteins to immobilized E. coli J5 lipid A was inhibited in a comparable fashion by long- or short-chain LPS or lipid A. The binding of both rBPI23 and nBPI55 was specific, saturable, and of high affinity, with an apparent Kd of approximately 2 to 5 nM for all ligands tested. These results demonstrate that BPI recognizes the highly conserved lipid A region of bacterial LPS via residues contained within the amino-terminal portion of the BPI molecule.
TL;DR: Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized and appears to interfere with cell division, since susceptible cells filamented when exposed to it.
Abstract: Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system.
TL;DR: The cloning, sequencing, and expression of a gene name cna, encoding a S. aureus collagen adhesin, is reported on, which reveals several structural characteristics similar to previously described Gram-positive bacterial cell surface proteins.
TL;DR: The two groups of pathogenic E. coli are both composed of a limited number of clones for which the O:K:H serotypes are excellent, although not faultless, markers.
Abstract: Serotyping of Escherichia coli is useful, but complex, with 173 O antigens, 80 K antigens, and 56 H antigens, which can all be subdivided into partial antigens. The O, K, and H antigens can be foun...
TL;DR: The inclusion body-associated proteins IbpA and ibpB were identified and isolated from the Kohara E. coli gene bank as mentioned in this paper, and they have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production.
Abstract: In Escherichia coli high-level production of some heterologous proteins (specifically, human prorenin, renin, and bovine insulin-like growth factor 2) resulted in the induction of two new E. coli heat shock proteins, both of which have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production. We named these inclusion body-associated proteins IbpA and IbpB. The coding sequences for IbpA and IbpB were identified and isolated from the Kohara E. coli gene bank. The genes for these proteins (ibpA and ibpB) are located at 82.5 min on the chromosome. Nucleotide sequencing of the two genes revealed that they are transcribed in the same direction and are separated by 110 bp. Putative Shine-Dalgarno sequences are located upstream from the initiation codons of both genes. A putative heat shock promoter is located upstream from ibpA, and a putative transcription terminator is located downstream from ibpB. A temperature upshift experiment in which we used a wild-type E. coli strain and an isogenic rpoH mutant strain indicated that a sigma 32-containing RNA polymerase is involved in the regulation of expression of these genes. There is 57.5% identity between the genes at the nucleotide level and 52.2% identity at the amino acid level. A search of the protein data bases showed that both of these 16-kDa proteins exhibit low levels of homology to low-molecular-weight heat shock proteins from eukaryotic species.
TL;DR: A model for the coupled action of cadA and cadB in uptake of lysine, the reduction of H+ concentration, and excretion of cadaverine is led to.
Abstract: Lysine decarboxylase of Escherichia coli has been the subject of enzymological studies, and the gene encoding lysine decarboxylase (cadA) and a regulatory gene (cadR) have been mapped. This enzyme is induced at low pH in the presence of lysine and achieves maximal level under anaerobic conditions. The induction of lysine decarboxylase increases the pH of the extracellular medium and provides a distinctive marker in tests of clinical strains. We report the sequence of the cad operon encoding lysine decarboxylase, a protein of 715 amino acids, and another protein, CadB, of 444 amino acids. The amino acid sequence of lysine decarboxylase showed high homology to that of the lysine decarboxylase of Hafnia alvei with less homology to the sequence of speC, which encodes the biosynthetic ornithine decarboxylase of E. coli. The cadA and cadB genes were separately cloned and placed under the control of lac and tac promoters, respectively, to facilitate independent study of their physiological effects. The cadB gene product had a mobility characteristic of a smaller protein on protein gels, analogous to that found for some other membrane proteins. The CadB sequence showed homology to that of ArcD of Pseudomonas aeruginosa, encoding an arginine/ornithine antiporter. Excretion studies of various strains, the coinduction of cadB and cadA, and the attractive physiological role for an antiport system led to a model for the coupled action of cadA and cadB in uptake of lysine, the reduction of H+ concentration, and excretion of cadaverine.
TL;DR: Results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step, and is susceptible to degradation by proteases in vivo and in vitro.
Abstract: Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm. The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space. It was susceptible to degradation by proteases in vivo and in vitro. The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues. PhoA synthesized in the mutants indeed lacked disulfide bridges. These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step.
TL;DR: This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli with high cell concentrations as well as high specific yields of recombinant product.
Abstract: Whereas cell concentrations of 5-10 grams dry cell weight per liter (gDCW/l) are typical of batch cultures, fed-batch techniques can be used to achieve concentrations greater than 50 gDCW/l. Feeding strategies for fed-batch cultures include feed-back control as well as pre-determined feeding profiles. The volumetric yield of recombinant products can be improved by controlling the specific growth rate and the substrate concentration. Furthermore, inhibitory by-product formation can be minimized in fed-batch cultures. This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli. The modes of nutrient feeding that have been employed are discussed, and the factors important in attaining high cell concentrations as well as high specific yields of recombinant product are described.
TL;DR: Results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.
Abstract: Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression. Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain. Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants. Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids. Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics. These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.
TL;DR: Although adhesion to intestinal epithelial cells and verotoxins are considered important virulence factors in the pathogenesis of the organism, more research is are necessary to determine the exact mechanism of pathogenicity.
TL;DR: YUH1 and UBP1, the genes for two ubiquitin-specific proteases of the yeast Saccharomyces cerevisiae, have been cloned previously and shown to encode nonhomologous proteins, but using an Escherichia coli-based genetic screen, two other yeast genes are isolated, named UBP2 and U BP3, indicating the presence of at least one more ubiquitIn-specific processing protease in S. cerevisia.
TL;DR: Subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilIS/C.
Abstract: We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C. acetobutylicum shuttle vector. Plasmid pFNK1 was used because shuttle vectors that function in E. coli were unable to electrotransform ATCC 824 unless they became deleted in the E. coli-plasmid regions. The difficulties with shuttle vectors that function in E. coli are probably due to the presence of a restriction endonuclease in ATCC 824. This endonuclease recognizes the sequence 5'-GCNGC-3', which is prevalent in E. coli plasmids but occurs infrequently in pFNK1 and C. acetobutylicum genes. Cloning of genes in C. acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.
TL;DR: The three-dimensional and clavulanate double-disk potentiation tests were compared as procedures for the detection of extended-spectrum beta-lactamase production in 32 strains of Escherichia coli and Klebsiella pneumoniae, offering the advantages of providing simultaneous information about both antibiotic susceptibility and extended- Spectrum beta- lactamases production.
Abstract: The three-dimensional and clavulanate double-disk potentiation tests were compared as procedures for the detection of extended-spectrum beta-lactamase production in 32 strains of Escherichia coli and Klebsiella pneumoniae, 31 of which produced TEM-1, TEM-2, TEM-3, TEM-4, TEM-5, TEM-7, TEM-8, TEM-9, TEM-10, TEM-12, TEM-101, SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, CAZ-2, MIR-1, or an unidentified extended-spectrum beta-lactamase with a pI of 5.95, with some strains producing multiple beta-lactamases. The three-dimensional test, which was performed in conjunction with a routine disk diffusion test, detected extended-spectrum beta-lactamase production in 26 of 28 (93%) of the strains that produced extended-spectrum beta-lactamases. The clavulanate double-disk potentiation test detected extended-spectrum beta-lactamases in only 22 of the 28 strains (79%) when it was performed as currently recommended. The three-dimensional test, when performed in conjunction with the disk diffusion test, offered the advantages of providing simultaneous information about both antibiotic susceptibility and extended-spectrum beta-lactamase production, coupled with a greater sensitivity and earlier detection of extended-spectrum beta-lactamases.
TL;DR: Nine H. pylori urease genes were identified and ureA, ureB,UreC, UreF, uREG, and UreH were shown to be required for ure enzyme expression in E. coli, as mutations in each of these genes led to negative phenotypes.
Abstract: Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.
TL;DR: A rapid and sensitive method for detection of Shiga-like toxin ( SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described.
Abstract: A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.
TL;DR: When proteoliposomes reconstituted from the purified OmpA, phospholipids, and lithium dodecyl sulfate were tested for permeability to small molecules by osmotic swelling, it was found that OMPA produced apparently nonspecific diffusion channels that allowed the penetration of various solutes.
TL;DR: The instability of SLT genes warrants direct screening methods for clinical material and the development of new culture methods to prevent the loss ofSLT genes.
Abstract: Forty-five consecutive patients with various gastrointestinal disorders were identified as having Shiga-like toxin (SLT)-producing Escherichia coli infections. This was shown by the cytotoxic effect of stool extracts in Vero cell cultures which was neutralizable by antibodies to SLTs and by isolation of E. coli that hybridized with DNA probes complementary to SLT-I and SLT-II sequences. When we tested the same strains for SLT genes after subcultivation, the isolates from 15 patients became negative by colony hybridization and polymerase chain reaction and failed to produce SLTs. The instability of SLT genes warrants direct screening methods for clinical material and the development of new culture methods to prevent the loss of SLT genes. Images