A novel genetic island of meningitic Escherichia coli K1 containing the ibeA invasion gene (GimA): functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells

The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process. Electronic Publication     

Nitrofurantoin-resistant mutants of Escherichia coli: isolation and mapping

Summary Mutants of Escherichia coli resistant to nitrofurantoin have been isolated. The mutations, designated nfnA and nfnB were introduced individually into a multiply auxotrophic E. coli F^– strain and mapped by conjugation and transduction. nfnA is located at 79.8 min and nfnB at 13.0 min on the E. coli chromosome.     

Genetic Mapping of Cold Resistance Gene of Escherichia coli

Using cold resistant mutants, MET1 and MET2, obtained from Escherichia coli K-12, genetic mapping of the cold resistance gene(s) of E. coli was performed by the conjugation and transduction techniques. The gene(s) was confirmed to be located close to trpB at 28 min (revised chromosome linkage map, 1983) on the E. coli chromosome.     

Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids

Summary The DNA polymerase induced by bacteriophage T7 is composed of a phage-specified subunit, the gene 5 protein, and a host-specified subunit, the 12,000 dalton thioredoxin of Escherichia coli. tsnC mutants of E. coli B (Chamberlin, 1974) have no detectable thioredoxin, and thus cannot support the growth of phage T7, although they are killed by phage infection. A mutant of E. coli K12 affecting thioredoxin has been isolated by a modification of the procedure used by Chamberlin (1974) to isolate tsnC mutants of E. coli B. The gene affecting thioredoxin has been designated trxA. This mutant, E. coli JM109, shows the TsnC phenotype in that it is killed by, but cannot support the growth of, bacteriophage T7. T7 DNA replication does not occur in mutantinfected cells. These phenotypic expressions of the tsnC mutation have enabled us to screen recombinants for the trxA allele in HfrxF^- crosses and F-ductants in episome transfer experiments. Extracts of transductants in generalized transduction by P1 phage were screened for their ability to complement partially purified phage T7 gene 5 protein to form T7 DNA polymerase. The trxA gene is located at 84 min on the E. coli linkage map, between uvrE and metE; trxA is 34% co-transducible with metE.     

Rapid Chromatographic Separation of Ribosomax Ribonucleic Acids

Reversed-phase chromatography has been applied to the rapid separation of E. coli 5S rRNA and has also been adapted for use on an analytical scale for the rapid (about 30 min) separation of small quantities (0.1 Ap₂₆₀ unit) of l6s and 23S rRNAs. Compared to other techniques, this nondestructive method is faster, more sensitive, and gives better resolution.     

Ribosomal protein modification in Escherichia coli

Summary A mutant of Escherichia coli K12 has been isolated which shows an alteration in the ribosomal protein S18. Genetic analyses have revealed that the mutation causing this alteration maps at 99.3 min of the E. coli genetic map, between dnaC and deo. This indicated that the mutation has occurred in a gene different from the structural gene for this protein which has been located at 94 min. From the N-terminal amino acid sequence analysis it is concluded that the mutation has resulted in loss of the N-terminal acetyl group of this protein. The gene which is affected in this mutant is termed rimI that most likely specifies an enzyme acetylating the N-terminal alanine of protein S18. The mutation does not affect the acetylation of two other ribosomal proteins, S5 and L12, both of which are known to be acetylated in wild-type E. coli K12.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

A MEMS based amperometric detector for E. Coli bacteria using self-assembled monolayers

We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA–rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.     

On the regulation of D-ribose metabolism in E. coli B-r. II. Chromosomal location and fine structure analysis of the D-ribose permease and D-ribokinase structural genes by P 1 transduction

Summary The genetic mapping and fine structure analysis of the d-ribose gene in Escherichia coli B/r has been studied. Findings indicate that the structural genes for the d-ribokinase and d-ribose permease map closely linked to the ara-leu region of the chromosome in contrast to their location in the isoleucine-valine region at 73.5 min in E. coli K12. Two polarity mutants, AB7 and AB36, were found to map at the left end of the d-ribokinase gene thus supporting the proposed d-ribokinase-d-ribose permease operon for the d-ribose catabolic enzymes in E. coli B/r.     

Cloning and Expression in Escherichia Coli of a Gene Encoding Superoxide Dismutase from Listeria Ivanovii

A chromosomal DNA fragment from the gram-positive bacterium Listeria ivanovii (ATCC 19119) encoding a superoxide dismutase (SOD) gene has been cloned in Escherichia coli QC779 (sodAsodB) using the plasmid vector pTZ19R. The DNA fragment inserted into the plasmid showed-high structural instability in E. coli QC779 (recA⁺). but turned out to be a stable 1.95 kbp DNA fragment when transformed into E. coli DHSa (recA-). The gene is expressed in both of these E. coli strains at high levels. Preliminary studies showed that the activity of the recombinant SOD within E. coli DHSα was up to 13-times the combined activity of both E. coli SODs. The recombinant SOD forms active hybrid SODS with both E. coli SODs in vivo.     

Effect of Fe/Al Hydroxides on Transport and Retention of Escherichia coli in Saturated Sand Media

The transport of bacteria has been investigated extensively using iron (Fe) (hydr)oxide-coated quartz. However, few studies have investigated the effects of aluminum (Al) (hydr)oxide on the transport of bacteria. In this study, column experiments were conducted to investigate the effects of Fe/Al hydroxides on the transport of Escherichia coli (E. coli) in saturated quartz sand at different pH levels, ionic strengths (IS), and ionic compositions. Fe/Al hydroxide coatings increased the positive charge of quartz, reduced the negative charge, shifted zeta potential in a positive direction, and thus enhanced the retention of E. coli on quartz. The retention of E. coli decreased with increasing pH and increased with increasing IS. These findings were consistent with the theoretical prediction of the Derjaguin, Landau, Verwey and Overbeek (DLVO) interaction energy. Calcium ions improved the retention of E. coli in the column. Since Al-hydroxide-coated quartz had a more positive charge, the retention of E. coli was higher in Al-hydroxide-coated quartz than in Fe-hydroxide-coated quartz. When compared with quartz alone, Fe/Al hydroxide coatings significantly reduced the transport of E. coli, and the inhibitory effect of Al hydroxide was greater than that of Fe hydroxide.     

Influence of light and natural microbiota of the Butrón river on E. coli survival

The survival of an E. coli strain in water samples from the Butrón river has been studied. The input of E. coli cells in the aquatic system breaks down the established balance among the components of the natural microbiota: E. coli becomes the object of the active protozoal predation whereas the autochtonous heterotrophic community become alternative preys. As a result of this new situation, the natural microbiota increases but returns to the initial values once the E. coli cells have been removed from the system. The effect of the temperature of incubation on the survival is exerted through the effect of this parameter on the predatory activity of the protozoa. Light has a lethal and direct action on the E. coli cells, the effect of this parameter is even superior to that of predation.     

Effects of 50 Hz rotating magnetic field on the viability of Escherichia coli and Staphylococcus aureus

This study presents results of research on the influence of rotating magnetic field (RMF) of the induction of 30?mT and the frequency of 50?Hz on the growth dynamics and cell metabolic activity of E. coli and S. aureus, depending on the exposure time. The studies showed that the RMF caused an increase in the growth and cell metabolic activity of all the analyzed bacterial strains, especially in the time interval t?=?30 to 150?min. However, it was also found that the optical density and cell metabolic activity after exposition to RMF were significantly higher in S. aureus cultures. In turn, the study of growth dynamics, revealed a rapid and a significant decrease in these values from t?=?90?min) in the case of E. coli samples. The obtained results prove that RMF (B?=?30?mT, f?=?50?Hz) has a stimulatory effect on the growth and metabolic activity of E. coli and S. aureus. Furthermore, taking into account the time of exposure, stronger influence of RMF on the viability was observed in S. aureus cultures, which may indicate that this effect depends on the shape of the exposed cells.     

Development of a multifunctional and efficient conjugal plasmid for use in Streptomyces spp

A plasmid, pGB112, has recently been developed to transfer DNA from Escherichia coli to Streptomyces spp via conjugation. This technique made use of (A) E. coli replicon, (B) ampicillin (amp) resistance gene for selection in E. coli and thiostrepton (tsr) resistance gene for selection in Streptomyces, (C) a fragment of SCP2* replicon, (D) a 2.6 kb fragment of tra-cassette which consists of pIJ101 transfer gene (tra) and two ermE promoters, (E) a 0.8 kb fragment of oriT of (IncP) RK2. The results showed that this plasmid was able to transfer plasmid DNA from E. coli to Streptomyces coelicolor via conjugation, and that it could also transfer DNA between Streptomyces strains. Since this plasmid has both pBR322 and SCP2* replicons, it may provide a novel and useful method for genetic operation in E. coli and Streptomyces.An erratum to this article can be found at     

A gene involved in the metabolic control of ppGpp synthesis

Summary A genetic locus has been identified which controls the basal synthesis of ppGpp in growing E. coli. Cells carrying a recessive allele of the relX gene have a very low concentration of ppGpp during balanced growth, and fail to accumulate ppGpp in response to carbon/energy source downshift. Moreover, the recessive relX allele renders the cells unable to grow at 42° C and, when coupled with relA, makes the cells sensitive to the presence of leucine in minimal medium. RelX is cotransduced with fuc and relA and located at approximately 59.4 min on the E. coli genetic map.     

The effect of manganese and iron on mediating resuscitation of lactic acid-injured Escherichia coli

Lactic acid can induce sublethal injury of E. coli through oxidative stress. In this study, we investigated changes in SOD activity, CAT activity, GSH production and ROS production during sublethal injury and resuscitation of E. coli. Then, the effect of manganese and iron during resuscitation were studied. Both cations (≥1 mmol l⁻¹) significantly promoted the resuscitation of sublethally injured E. coli induced by lactic acid and shortened the repair time (P < 0·05). Conversely, addition of N,N,N′,N′-tetrakis (2-pyridylmethyl) which is a metal chelator extended the repair time. Compared with minA, manganese and iron significantly improved SOD activity at 40, 80 and 120 min and decreased ROS production at 40 and 80 min, thereby recovering injured E. coli quickly (P < 0·05). The deletion of sodA encoding Mn-SOD, sodB encoding Fe-SOD or gshA/gshB encoding GSH significantly strengthened sublethal injury and extended the repair time (P < 0·05). It meant these genes-related oxidative stress played important roles in the acid resistance of E. coli and recovery of sublethal injury. Therefore, manganese and iron can promote the recovery of lactic-injured E. coli by the way of increasing SOD activity, scavenging ROS, and relieving oxidative stress.     

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains.The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.     

Characterization of <Emphasis Type="Italic">Escherichia coli</Emphasis> EutD: a phosphotransacetylase of the ethanolamine operon

The Escherichia coli genes pta and eutD encode proteins containing the phosphate-acetyltransferase domain. EutD is composed only by this domain and belongs to the ethanolamine operon. This enzyme has not been characterized yet, and its relationship to the multimodular E. coli phosphotransacetylase (Pta) remains unclear. In the present work, a detailed characterization of EutD from E. coli (EcEutD) was performed. The enzyme is a more efficient phosphotransacetylase than E. coli Pta (EcPta) in catalyzing its reaction in either direction and assembles as a dimer, being differentially modulated by EcPta effectors. When comparing EutD and Pta, both from E. coli, certain divergent regions of the primary structure responsible for their unique properties can be found. The growth on acetate of the E. coli pta acs double-mutant strain, was complemented by either introducing EcEutD or by inducing the eut operon with ethanolamine. In this case, the expression of a phosphotransacetylase different from Pta was confirmed by activity assays. Overall, the results indicate that EcEutD and Pta, although able to catalyse the same reaction, display differential efficiency and regulation, and also differ in the induction of their expression. However, under certain growth conditions, they can fulfil equal roles in E. coli metabolism.     

Chemiluminescence detection of Escherichia coli in fresh produce obtained from different sources

A chemiluminescence‐based assay is developed for the rapid detection of Escherichia coli in fresh produce. The assay was based on the reaction of β‐galactosidase enzyme from E. coli with a phenylgalactosidase‐substituted dioxetane substrate. Light emitted from the reaction was measured in a luminometer and data correlated with counts of E. coli enumerated on sorbitol–MacConkey agar plates. A strain of E. coli O157:H7 was used to inoculate samples of fresh produce to differentiate the inoculum from the natural E. coli potentially present on the produce. Fresh market samples were tested for generic E. coli and E. coli O157:H7. Signi?cant differences in light emission were found in samples with high initial E. coli counts when market samples were compared to respective heat‐treated samples. The assay was able to detect E. coli in all produce tested, particularly at higher contamination or inoculation levels. The sensitivity of the assay ranged between 10²–10⁵ CFU within 30 min. The chemiluminescence assay provides a simple and rapid method for detection of viable E. coli, an important step towards enhancing food safety. Copyright © 2004 John Wiley & Sons, Ltd.     

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation

Summary A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli SecA gene as a probe. The deduced amino acid sequence showed 58% identity to the aminoterminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secA ^ts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.