Showing papers on "Escherichia coli published in 2000"
TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
14,389 citations
TL;DR: The existence of elements of silver and sulfur in the electron-dense granules and cytoplasm detected by X-ray microanalysis suggested the antibacterial mechanism of silver: DNA lost its replication ability and the protein became inactivated after Ag(+) treatment.
Abstract: To investigate the mechanism of inhibition of silver ions on microorganisms, two strains of bacteria, namely Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), were treated with AgNO(3) and studied using combined electron microscopy and X-ray microanalysis. Similar morphological changes occurred in both E. coli and S. aureus cells after Ag(+) treatment. The cytoplasm membrane detached from the cell wall. A remarkable electron-light region appeared in the center of the cells, which contained condensed deoxyribonucleic acid (DNA) molecules. There are many small electron-dense granules either surrounding the cell wall or depositing inside the cells. The existence of elements of silver and sulfur in the electron-dense granules and cytoplasm detected by X-ray microanalysis suggested the antibacterial mechanism of silver: DNA lost its replication ability and the protein became inactivated after Ag(+) treatment. The slighter morphological changes of S. aureus compared with E. coli recommended a defense system of S. aureus against the inhibitory effects of Ag(+) ions.
3,811 citations
TL;DR: A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA using a defective lambda prophage, which will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.
Abstract: A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.
1,790 citations
TL;DR: These findings provide novel insights into the VFs of extraintestinal pathogenic E. coli and demonstrate the new PCR assay's utility for molecular epidemiological studies.
Abstract: Among 75 urosepsis isolates of Escherichia coli, 29 virulence factor (VF) genes were detected by use of a novel polymerase chain reaction (PCR) assay. Compared with probe hybridization, the PCR assay's specificity was 100% and sensitivity 97.1%. fyuA (yersiniabactin: overall prevalence, 93%), traT (serum resistance, 68%), and a pathogenicity-associated island marker (71%) occurred in most strains from both compromised and noncompromised hosts. Present in <20% of strains each were sfaS, focG (F1C fimbriae), afa/dra, bmaE (M fimbriae), gafD (G fimbriae), cnf1, cdtB (cytolethal distending toxin), cvaC (colicin V), and ibeA (invasion of brain endothelium). Different VFs were variously confined to virulence-associated phylogenetic group B2 (as defined by multilocus enzyme electrophoresis); concentrated in group B2, but with spread beyond; or concentrated outside of group B2. These findings provide novel insights into the VFs of extraintestinal pathogenic E. coli and demonstrate the new PCR assay's utility for molecular epidemiological studies.
1,225 citations
TL;DR: The construction of strains carrying mutations in multiple genes is helping to elucidate the different roles of glutathione and thioredoxin, and studies with such strains have recently revealed that these two reduction systems modulate the activities of the E. coli OxyR and SoxR and the S. cerevisiae Yap1p transcriptional regulators of the adaptive responses to oxidative stress.
Abstract: The glutathione- and thioredoxin-dependent reduction systems are responsible for maintaining the reduced environment of the Escherichia coli and Saccharomyces cerevisiae cytosol. Here we examine the roles of these two cellular reduction systems in the bacterial and yeast defenses against oxidative stress. The transcription of a subset of the genes encoding glutathione biosynthetic enzymes, glutathione reductases, glutaredoxins, thioredoxins, and thioredoxin reductases, as well as glutathione- and thioredoxin-dependent peroxidases is clearly induced by oxidative stress in both organisms. However, only some strains carrying mutations in single genes are hypersensitive to oxidants. This is due, in part, to the redundant effects of the gene products and the overlap between the two reduction systems. The construction of strains carrying mutations in multiple genes is helping to elucidate the different roles of glutathione and thioredoxin, and studies with such strains have recently revealed that these two reduction systems modulate the activities of the E. coli OxyR and SoxR and the S. cerevisiae Yap1p transcriptional regulators of the adaptive responses to oxidative stress.
674 citations
TL;DR: Treatment of human STEC infection with bacteriophage-inducing antibiotics, such as fluoroquinolones, may have significant adverse clinical consequences and that fluoroquolone antibiotics may enhance the movement of virulence factors in vivo.
Abstract: Shiga toxin-producing Escherichia coli (STEC) cause significant disease; treatment is supportive and antibiotic use is controversial. Ciprofloxacin but not fosfomycin causes Shiga toxin-encoding bacteriophage induction and enhanced Shiga toxin (Stx) production from E. coli O157:H7 in vitro. The potential clinical relevance of this was examined in mice colonized with E. coli O157:H7 and given either ciprofloxacin or fosfomycin. Both antibiotics caused a reduction in fecal STEC. However, animals treated with ciprofloxacin had a marked increase in free fecal Stx, associated with death in two-thirds of the mice, whereas fosfomycin did not. Experiments that used a kanamycin-marked Stx2 prophage demonstrated that ciprofloxacin, but not fosfomycin, caused enhanced intraintestinal transfer of Stx2 prophage from one E. coli to another. These observations suggest that treatment of human STEC infection with bacteriophage-inducing antibiotics, such as fluoroquinolones, may have significant adverse clinical consequences and that fluoroquinolone antibiotics may enhance the movement of virulence factors in vivo.
564 citations
TL;DR: Phylogenetic analysis reveals that old lineages of E. coli have acquired the same virulence factors in parallel, including a pathogenicity island involved in intestinal adhesion, a plasmid-borne haemolysin, and phage-encoded Shiga toxins, indicating that natural selection has favoured an ordered acquisition of genes and the progressive build-up of molecular mechanisms that increase virulence.
Abstract: The mechanisms underlying the evolution and emergence of new bacterial pathogens are not well understood. To elucidate the evolution of pathogenic Escherichia coli strains, here we sequenced seven housekeeping genes to build a phylogenetic tree and trace the history of the acquisition of virulence genes. Compatibility analysis indicates that more than 70% of the informative sites agree with a single phylogeny, suggesting that recombination has not completely obscured the remnants of ancestral chromosomes1,2,3. On the basis of the rate of synonymous substitution for E. coli and Salmonella enterica (4.7 × 10-9 per site per year3), the radiation of clones began about 9 million years ago and the highly virulent pathogen responsible for epidemics of food poisoning, E. coli O157:H7, separated from a common ancestor of E. coli K-12 as long as 4.5 million years ago. Phylogenetic analysis reveals that old lineages of E. coli have acquired the same virulence factors in parallel, including a pathogenicity island involved in intestinal adhesion, a plasmid-borne haemolysin, and phage-encoded Shiga toxins. Such parallel evolution indicates that natural selection has favoured an ordered acquisition of genes and the progressive build-up of molecular mechanisms that increase virulence.
506 citations
TL;DR: The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases.
Abstract: The copA gene product, a putative copper-translocating P-type ATPase, has been shown to be involved in copper resistance in Escherichia coli. The copA gene was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain was more sensitive to copper salts but not to salts of other metals, suggesting a role in copper homeostasis. The copper-sensitive phenotype could be rescued by complementation by a plasmid carrying copA from E. coli or copB from Enterococcus hirae. Expression of copA was induced by salts of copper or silver but not zinc or cobalt. Everted membrane vesicles from cells expressing copA exhibited ATP-coupled accumulation of copper, presumably as Cu(I). The results indicate that CopA is a Cu(I)-translocating efflux pump that is similar to the copper pumps related to Menkes and Wilson diseases and provides a useful prokaryotic model for these human diseases.
493 citations
TL;DR: This research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis and shown the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.
Abstract: Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.
474 citations
TL;DR: A two-step technology takes advantage of an Escherichia coli strain expressing the phage lambda Red functions and enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%.
Abstract: The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage λ Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements.
456 citations
TL;DR: The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described, and has been associated with iron transport and metabolism.
Abstract: In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E. coli. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.
TL;DR: It is found that neutrophil elastase degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria, defining a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense.
Abstract: In determining the mechanism of neutrophil elastase (NE)-mediated killing of Escherichia coli, we found that NE degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria. NE killed wild-type, but not OmpA-deficient, E. coli. Also, whereas NE-deficient mice had impaired survival in response to E. coli sepsis, as compared to wild-type mice, the presence or absence of NE had no influence on survival in response to sepsis that had been induced with OmpA-deficient E. coli. These findings define a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense.
TL;DR: Nearly 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between Ntr C and final sigma(70)-dependent promoters.
Abstract: Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, ≈2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and σ70-dependent promoters.
TL;DR: It is concluded that Iha is a novel bacterial adherence-conferring protein and is contained within an E. coli chromosomal island of conserved structure.
Abstract: The mechanisms used by Shiga toxin (Stx)-producing Escherichia coli to adhere to epithelial cells are incompletely understood. Two cosmids from an E. coli O157:H7 DNA library contain an adherence-conferring chromosomal gene encoding a protein similar to iron-regulated gene A (IrgA) of Vibrio cholerae (M. B. Goldberg, S. A. Boyko, J. R. Butterton, J. A. Stoebner, S. M. Payne, and S. B. Calderwood, Mol. Microbiol. 6:2407-2418, 1992). We have termed the product of this gene the IrgA homologue adhesin (Iha), which is encoded by iha. Iha is 67 kDa in E. coli O157:H7 and 78 kDa in laboratory E. coli and is structurally unlike other known adhesins. DNA adjacent to iha contains tellurite resistance loci and is conserved in structure in distantly related pathogenic E. coli, but it is absent from nontoxigenic E. coli O55:H7, sorbitol-fermenting Stx-producing E. coli O157:H-, and laboratory E. coli. We have termed this region the tellurite resistance- and adherence-conferring island. We conclude that Iha is a novel bacterial adherence-conferring protein and is contained within an E. coli chromosomal island of conserved structure. Pathogenic E. coli O157:H7 has only recently acquired this island.
TL;DR: It is reported that Ag43 contributes to E. coli biofilm formation in glucose‐minimal medium, but not in Luria–Bertani broth, and it is shown that flagellar‐mediated motility is required for biofilm Formation in both rich and minimal environments.
Abstract: Transcription of the agn43 locus, which specifies an outer membrane protein of Escherichia coli, is regulated in a phase-variable fashion by the OxyR-DNA binding protein and Dam methylase. Despite its well-characterized regulation, the function of Ag43 has remained elusive until now. Previous studies indicated that Ag43 mediates autoaggregation of certain strains of E. coli in liquid culture. Given this phenotype, we examined the role of Ag43 in biofilm formation. Here, we report that Ag43 contributes to E. coli biofilm formation in glucose-minimal medium, but not in Luria-Bertani broth. In addition, we show that flagellar-mediated motility is required for biofilm formation in both rich and minimal environments. Altogether, our results suggest that E. coli uses both common and specific gene sets for the development of biofilms under various growth conditions.
TL;DR: The most important factors for E. coli 0157 are the production of Shiga toxin 2 and the adhesin intimin and the role of some of the other virulence factors, such as enterohaemolysin, a serine protease (EspP) and a catalase/peroxidase (Katp), in infection may be low.
Abstract: Shiga toxin-producing Escherichia coli 0157 are important enteropathogens causing outbreaks of haemorrhagic colitis and haemolytic uraemic syndrome. Strains of E. coli belonging to other serogroups also produce Shiga toxins but are less frequently isolated from cases of diarrhoeal illness despite humans having greater exposure to these organisms in food and the environment. It is generally considered that E. coli O157 is more virulent than these other Shiga toxin-producing E. coli. The question of which factors make toxigenic E. coli 0157 more virulent is unanswered. In this review the various virulence properties of E. coli 0157 and their incidence in non-0157 Shiga toxin-producing E. coli are described. The most important factors for E. coli 0157 are the production of Shiga toxin 2 and the adhesin intimin. The role of some of the other virulence factors, such as enterohaemolysin, a serine protease (EspP) and a catalase/peroxidase (Katp), in infection may be low. Uncharacterized virulence properties such as a clostridial-like toxin and haemoglobin uptake require further study and it is likely that other virulence properties remain to be discovered.
TL;DR: SOS-mediated induction of toxin synthesis appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene, and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease.
Abstract: Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene. Phage production is linked to induction of the bacterial SOS response, a ubiquitous response to DNA damage. SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression in studies of their effects on a reporter STEC strain carrying a chromosomebased stx2::lacZ transcriptional fusion. At antimicrobial levels above those required to inhibit bacterial replication, these agents are potent inducers (up to 140-fold) of the transcription of type 2 Shiga toxin genes (stx2); therefore, they should be avoided in treating patients with potential or confirmed STEC infections. Other agents (20 studied) and incubation conditions produced significant but less striking effects on stx2 transcription; positive and negative influences were observed. SOS-mediated induction of toxin synthesis also provides a mechanism that could exacerbate STEC infections and increase dissemination of stx genes. These features and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease.
TL;DR: Two chromosomal genes are identified from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions that is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco.
Abstract: Using a genetic screen we have identified two chromosomal genes, cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the pco operon of E. coli; CopR and CopS from the cop operon, which provides copper resistance to Pseudomonas syringae; and SilR and SilS from the sil locus, which provides silver ion resistance to Salmonella enterica serovar Typhimurium. The genes cusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC (ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. The cus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusC and additional downstream genes are homologous to known metal ion antiporters.
TL;DR: The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx 2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.
Abstract: We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx(2f). This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx(2f) gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.
TL;DR: Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones and revealed that one recombinant strain harbored a lipase and the other three contained esterases.
Abstract: Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.
TL;DR: The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E. coli without prior cloning of the gene of interest.
TL;DR: It is demonstrated that vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109 and can deliver antibiotic resistance.
Abstract: Many gram-negative bacteria produce membrane vesicles, suggesting that vesicle production is not purposeless; indeed, studies during the last two decades have presented strong evidence supporting the importance of vesicles. Typical vesicles released from the surfaces of gram-negative bacteria are 50 to 250 nm, spherical, and made up of outer membrane and encapsulated periplasmic components (4, 26). Vesicle components include outer membrane proteins, lipopolysaccharide, periplasmic proteins, phospholipids, DNA, and RNA (9, 12, 15, 22, 34, 40). Vesicles from gram-negative bacteria were reported to fuse to both gram-positive and gram-negative bacteria and in some instances to promote lysis of the target cell (28). Moreover, vesicles may function as an alternative secretory pathway (3, 23) and promote adherence of the parent cell to host cells (17, 32). By virtue of their small size, bilayer protecting envelope, and ability to integrate into the membranes of foreign bacteria and to adhere to or be engulfed by eukaryotic cells, a potential role of vesicles in delivery of virulence factors, including enzymes and toxins, is not unlikely (23). In fact, virulence factors associated with the parent strain, including proteases, phospholipases, autolysin, hemolysins, and Shiga toxins, have been isolated from vesicles (3, 22, 26, 28).
Aside from toxic compounds, DNA has also been isolated from vesicles. Vesicles produced by Pseudomonas aeruginosa were reported to contain DNA (22). Vesicles released by Neisseria gonorrhoeae harbor both linear and circular DNA, including 4.2- and 7.1-kb plasmids (12). Chromosomal and bacteriophage-associated virulence genes were detected in Escherichia coli O157:H7 vesicles (26). Moreover, this research demonstrated that DNA was protected from digestion by DNase, suggesting that DNA is packaged within vesicles (26).
Bacterial evolution often proceeds by horizontal gene transfer between different genera and species (1, 7). Antibiotic resistance genes and pathogenicity islands have been acquired by a variety of pathogens, including E. coli, Salmonella enterica serovar Typhimurium, Yersinia pestis, Dichelobacter nodosis, and Helicobacter pylori (19). Virulence factors contributing to the pathogenicity of E. coli O157:H7, including Shiga toxins (45, 46) and intimin (31, 44), are encoded on pathogenicity islands in the O157 chromosome and are thought to have been acquired by horizontal transfer. Results of previous studies suggest that vesicles may be involved in the transfer of genetic material among similar bacterial species (8, 12, 26). The hypothesis has been put forth that vesicles influence antibiotic resistance in other bacteria in two ways: by physical dissemination of preformed antibiotic-inactivating enzymes into the recipient periplasm and by delivery of antibiotic resistance plasmids (3, 12). Competent Haemophilus influenzae produces vesicles which are released into the medium when cells are returned to normal growth conditions or a noncompetent state (8). Specific DNA-binding peptides were reported to be present on the surfaces of H. influenzae vesicles (24, 25) and to be associated with vesicles from N. gonorrhoeae (11).
Previously, it was reported that vesicles released by E. coli O157:H7 into culture medium contain virulence genes and Shiga toxin (26). In the present study, we demonstrate that E. coli O157:H7 vesicles mediate the transfer of virulence genes, which are subsequently expressed by recipient enteric bacteria. Moreover, the origin of the DNA in E. coli O157:H7 vesicles is elucidated. Observations show that in addition to bacteriophage-associated genes, E. coli O157:H7 vesicles contain plasmids and fragments of chromosomal DNA.
TL;DR: The cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp.
Abstract: NRAMPs (natural resistance-associated macrophage proteins) have been characterized in mammals as divalent transition metal transporters involved in iron metabolism and host resistance to certain pathogens. The mechanism of pathogen resistance is proposed to involve sequestration of Fe2+ and Mn2+, cofactors of both prokaryotic and eukaryotic catalases and superoxide dismutases, not only to protect the macrophage against its own generation of reactive oxygen species, but to deny the cations to the pathogen for synthesis of its protective enzymes. NRAMP homologues are also present in bacteria. We report the cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp. typhimurium, and the cloning of two distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia. The genes are designated mntH because the two enterobacterial NRAMPs encode H+-stimulated, highly selective manganese(II) transport systems, accounting for all Mn2+ uptake in each species under the conditions tested. For S. typhimurium MntH, the Km for 54Mn2+ (≈ 0.1 µM) was pH independent, but maximal uptake increased as pH decreased. Monovalent cations, osmotic strength, Mg2+ and Ca2+ did not inhibit 54Mn2+ uptake. Ni2+, Cu2+ and Zn2+ inhibited uptake with Kis greater than 100 µM, Co2+ with a Ki of 20 µM and Fe2+ with a Ki that decreased from 100 µM at pH 7.6 to 10 µM at pH 5.5. Fe3+ and Pb2+ inhibited weakly, exhibiting Kis of 50 µM, while Cd2+ was a potent inhibitor with a Ki of about 1 µM. E. coli MntH had a similar inhibition profile, except that Kis were three- to 10-fold higher. Both S. typhimurium and E. coli MntH also transport 55Fe2+ however, the Kms are equivalent to the Kis for Fe2+ inhibition of Mn2+ uptake, and are thus too high to be physiologically relevant. In both S. typhimurium and E. coli, mntH::lacZ constructs were strongly induced by hydrogen peroxide, weakly induced by EDTA and unresponsive to paraquat, consistent with the presence of Fur and OxyR binding sites in the promoters. Strains overexpressing mntH were more susceptible to growth inhibition by Mn2+ and Cd2+ than wild type, and strains lacking a functional mntH gene were more susceptible to killing by hydrogen peroxide. In S. typhimurium strain SL1344, mntH mutants showed no defect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cells; however, expression of mntH::lacZ was induced severalfold by 3 h after invasion of the macrophages. S. typhimurium mntH mutants showed only a slight attenuation of virulence in BALB/c mice. Thus, the NRAMP Mn2+ transporter MntH and Mn2+ play a role in bacterial response to reactive oxygen species and possibly have a role in pathogenesis.
TL;DR: To the authors' knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.
Abstract: The mechanisms responsible for macrolide resistance in Streptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A), erm(A), erm(B), erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids ((63)KPWRQKGTGRAR(74)) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.
TL;DR: Data indicated that OTAP-92 possesses a unique structural motif similar to the insect defensins, capable of killing Gram-negative bacteria by crossing the OM by a self-promoted uptake and cause damage to the biological function of cytoplasmic membrane.
TL;DR: Over forty years of research on the L-arabinose operon of Escherichia coli have provided insights into the mechanism of positive regulation of gene activity and the mechanism by which the regulatory protein changes its DNA-binding properties in response to the presence of arabinose.
TL;DR: Radioactivity from [2-(14)C]2C-methyl-D-erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis.
Abstract: In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP, which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using (13)C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn(2+) or Mg(2+) but no other cofactors. Radioactivity from [2-(14)C]2C-methyl-D-erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol into 2C-methyl-D-erythritol 3,4-cyclophosphate. This compound does not serve as substrate for the formation of carotenoids by isolated chromoplasts and is assumed to be an in vitro product without metabolic relevance.
TL;DR: A set of shuttle vectors, able to replicate in Escherichia coli and in gram-positive bacteria, containing a nisin-inducible promoter (PnisA) and genes encoding NisR and NisK, the two-component signaling mechanism for activating transcription from PnisA in the presence of nisin were constructed.
TL;DR: Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containingcis-prolines, concluding that the effect of FpA is independent of its PPIase activity.
TL;DR: Results demonstrate that thetsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.
Abstract: The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain χ7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with χ7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain χ7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain χ7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.