Showing papers on "Escherichia coli published in 2009"
TL;DR: The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells while transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.
894 citations
TL;DR: To investigate antibacterial activities of zinc oxide nanoparticles (ZnO NP) and their mode of action against an important foodborne pathogen, Escherichia coli O157:H7.
Abstract: Aims: To investigate antibacterial activities of zinc oxide nanoparticles (ZnO NP) and their mode of action against an important foodborne pathogen, Escherichia coli O157:H7.
Methods and Results: ZnO NP with sizes of 70 nm and concentrations of 0, 3, 6 and 12 mmol l−1 and NP-free solutions were used in antimicrobial tests against E. coli O157:H7. ZnO NP showed increasing inhibitory effects on the growth of E. coli O157:H7 as the concentrations of ZnO NP increased. A complete inhibition of microbial growth was achieved at the concentration level of 12 mmol l−1 or higher. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy were used to characterize the changes of morphology and cellular compositions of bacterial cells treated with ZnO NP and study the mode of action of ZnO NP against E. coli O157:H7. The intensity of lipid and protein bands in the Raman spectra of bacterial cells increased after exposure to ZnO NP, while no significant changes in nucleic acid bands were observed.
Conclusions: ZnO NP were found to have antibacterial activity against E. coli O157:H7. The inhibitory effects increase as the concentration of ZnO NP increased. Results indicate that ZnO NP may distort and damage bacterial cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells.
Significance and Impact of the Study: These results suggest that ZnO NP could potentially be used as an effective antibacterial agent to protect agricultural and food safety.
735 citations
TL;DR: It is demonstrated that, during growth, Pseudomonas aeruginosa produces an organic compound, identified as cis-2-decenoic acid, which is capable of inducing the dispersion of established biofilms and of inhibiting biofilm development.
Abstract: It is well established that in nature, bacteria are found primarily as residents of surface-associated communities called biofilms. These structures form in a sequential process initiated by attachment of cells to a surface, followed by the formation of matrix-enmeshed microcolonies, and culminating in dispersion of the bacteria from the mature biofilm. In the present study, we have demonstrated that, during growth, Pseudomonas aeruginosa produces an organic compound we have identified as cis-2-decenoic acid, which is capable of inducing the dispersion of established biofilms and of inhibiting biofilm development. When added exogenously to P. aeruginosa PAO1 biofilms at a native concentration of 2.5 nM, cis-2-decenoic acid was shown to induce the dispersion of biofilm microcolonies. This molecule was also shown to induce dispersion of biofilms, formed by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Bacillus subtilis, Staphylococcus aureus, and the yeast Candida albicans. Active at nanomolar concentrations, cis-2-decenoic acid appears to be functionally and structurally related to the class of short-chain fatty acid signaling molecules such as diffusible signal factor, which act as cell-to-cell communication molecules in bacteria and fungi.
540 citations
TL;DR: Findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means.
Abstract: Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance. We investigated persistence to the fluoroquinolone ciprofloxacin in Escherichia coli. Our data show that a majority of persisters to ciprofloxacin were formed upon exposure to the antibiotic, in a manner dependent on the SOS gene network. These findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means. SOS-induced persistence is a novel mechanism by which cells can counteract DNA damage and promote survival to fluoroquinolones. This unique survival mechanism may be an important factor influencing the outcome of antibiotic therapy in vivo.
461 citations
TL;DR: The evolution of these virulence plasmids and the implications of their acquisition by E. coli are now better understood and appreciated, with the available plasmid genomic sequences for several E. Escherichia coli pathotypes being compared in an effort to understand the evolution and define their core and accessory components.
Abstract: Bacterial plasmids are self-replicating, extrachromosomal elements that are key agents of change in microbial populations. They promote the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to antimicrobial agents, and metabolism of rare substances. Escherichia coli, perhaps the most studied of microorganisms, has been found to possess a variety of plasmid types. Included among these are plasmids associated with virulence. Several types of E. coli virulence plasmids exist, including those essential for the virulence of enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and extraintestinal pathogenic E. coli. Despite their diversity, these plasmids belong to a few plasmid backbones that present themselves in a conserved and syntenic manner. Thanks to some recent research, including sequence analysis of several representative plasmid genomes and molecular pathogenesis studies, the evolution of these virulence plasmids and the implications of their acquisition by E. coli are now better understood and appreciated. Here, work involving each of the E. coli virulence plasmid types is summarized, with the available plasmid genomic sequences for several E. coli pathotypes being compared in an effort to understand the evolution of these plasmid types and define their core and accessory components.
406 citations
TL;DR: A genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains, finds many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EH EC.
Abstract: Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5–5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.
348 citations
TL;DR: Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. bacteria pan-genome.
345 citations
TL;DR: The genome analysis revealed the entire gene repertoire related to E2348/69 virulence, and provided the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context.
Abstract: Enteropathogenic Escherichia coli (EPEC) was the first pathovar of E. coli to be implicated in human disease; however, no EPEC strain has been fully sequenced until now. Strain E2348/69 (serotype O127:H6 belonging to E. coli phylogroup B2) has been used worldwide as a prototype strain to study EPEC biology, genetics, and virulence. Studies of E2348/69 led to the discovery of the locus of enterocyte effacement-encoded type III secretion system (T3SS) and its cognate effectors, which play a vital role in attaching and effacing lesion formation on gut epithelial cells. In this study, we determined the complete genomic sequence of E2348/69 and performed genomic comparisons with other important E. coli strains. We identified 424 E2348/69-specific genes, most of which are carried on mobile genetic elements, and a number of genetic traits specifically conserved in phylogroup B2 strains irrespective of their pathotypes, including the absence of the ETT2-related T3SS, which is present in E. coli strains belonging to all other phylogroups. The genome analysis revealed the entire gene repertoire related to E2348/69 virulence. Interestingly, E2348/69 contains only 21 intact T3SS effector genes, all of which are carried on prophages and integrative elements, compared to over 50 effector genes in enterohemorrhagic E. coli O157. As E2348/69 is the most-studied pathogenic E. coli strain, this study provides a genomic context for the vast amount of existing experimental data. The unexpected simplicity of the E2348/69 T3SS provides the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context.
325 citations
TL;DR: In this paper, a review of toxicity and tolerance of organic acids, furan derivatives, and phenolic compounds with a specific focus on the important industrial organism Escherichia coli is presented.
Abstract: The sustainable production of biofuels will require the efficient utilization of lignocellulosic biomass. A key barrier involves the creation of growth-inhibitory compounds by chemical pretreatment steps, which ultimately reduce the efficiency of fermentative microbial biocatalysts. The primary toxins include organic acids, furan derivatives, and phenolic compounds. Weak acids enter the cell and dissociate, resulting in a drop in intracellular pH as well as various anion-specific effects on metabolism. Furan derivatives, dehydration products of hexose and pentose sugars, have been shown to hinder fermentative enzyme function. Phenolic compounds, formed from lignin, can disrupt membranes and are hypothesized to interfere with the function of intracellular hydrophobic targets. This review covers mechanisms of toxicity and tolerance for these compounds with a specific focus on the important industrial organism Escherichia coli. Recent efforts to engineer E. coli for improved tolerance to these toxins are also discussed.
324 citations
TL;DR: Two high‐cell‐density bacterial expression methods are explored, including an autoinduction introduced by Studier recently and a high‐cells‐density IPTG‐induction method described in this study, to achieve a cell‐density OD600 of 10–20 in the normal laboratory setting using a regular incubator shaker, allowing for consistently obtaining such a high yield of recombinant proteins using E. coli expression.
Abstract: The gram-negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high-level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields of the foreign eukaryotic proteins. We have been working on optimizations of bacterial expression conditions and media with a focus on achieving very high cell density for high-level production of eukaryotic proteins. Two high-cell-density bacterial expression methods have been explored, including an autoinduction introduced by Studier (Protein Expr Purif 2005;41:207–234) recently and a high-cell-density IPTG-induction method described in this study, to achieve a cell-density OD600 of 10–20 in the normal laboratory setting using a regular incubator shaker. Several practical protocols have been implemented with these high-cell-density expression methods to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14–25 mg of NMR triple-labeled proteins and 17–34 mg of unlabeled proteins from a 50-mL cell culture for all seven proteins we tested. Such a high protein yield used the same DNA constructs, bacterial strains, and a regular incubator shaker and no fermentor is necessary. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression.
TL;DR: Chemically inducible chromosomal evolution (CIChE), a plasmid-free, high gene copy expression system for engineering Escherichia coli, which improved genetic stability approximately tenfold and growth phase–specific productivity approximately fourfold for a strain producing the high metabolic burden–biopolymer poly-3-hydroxybutyrate.
Abstract: Engineering robust microbes for the biotech industry typically requires high-level, genetically stable expression of heterologous genes and pathways. Although plasmids have been used for this task, fundamental issues concerning their genetic stability have not been adequately addressed. Here we describe chemically inducible chromosomal evolution (CIChE), a plasmid-free, high gene copy expression system for engineering Escherichia coli. CIChE uses E. coli recA homologous recombination to evolve a chromosome with approximately 40 consecutive copies of a recombinant pathway. Pathway copy number is stabilized by recA knockout, and the resulting engineered strain requires no selection markers and is unaffected by plasmid instabilities. Comparison of CIChE-engineered strains with equivalent plasmids revealed that CIChE improved genetic stability approximately tenfold and growth phase-specific productivity approximately fourfold for a strain producing the high metabolic burden-biopolymer poly-3-hydroxybutyrate. We also increased the yield of the nutraceutical lycopene by 60%. CIChE should be applicable in many organisms, as it only requires having targeted genomic integration methods and a recA homolog.
TL;DR: It is likely that the observed resistance of bioFilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilm and that the production and resistance of the subpopulation were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.
Abstract: Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.
TL;DR: It is suggested that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract.
Abstract: Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.
TL;DR: The use of various metabolic engineering strategies to redirect the carbon flux inside E. coli to pathways responsible for the generation of malonyl-CoA led to the creation of an E. Escherichia coli strain with 15-fold elevated cellular malony lCoA level, which should be highly useful for improved production of important natural products where the cellular maloneyl- CoA level is rate-limiting.
TL;DR: HicB neutralizes HicA and therefore functions as an antitoxin, and as with other antitoxins, HicB could resuscitate cells inhibited by HICA, indicating that ectopic production ofHicA induces a bacteriostatic rather than a bactericidal condition.
Abstract: Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.
TL;DR: Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence.
Abstract: Although research has increasingly focused on the pathogenesis of avian pathogenic Escherichia coli (APEC) infections and the “APEC pathotype” itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (n = 121) with nonoutbreak strains, including fecal E. coli strains from clinically healthy chickens (n = 211) and strains from their environment (n = 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic E. coli (ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal E. coli strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.
TL;DR: A novel quantitative metabolomic approach based on stable isotope dilution is developed to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease and argues that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens.
Abstract: Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.
TL;DR: It is found that adding r-EPS significantly repressed biofilm formation by affecting genes related to curli production (crl, csgA, and csgB) and chemotaxis (cheY) in transcriptome analysis and could lead to the development of novel food-grade adjuncts for microbial biofilm control.
TL;DR: This study identified a novel class D β-lactamase involved in carbapenem resistance in A. baumannii, the first member of a novel subgroup of CHDLs whose prevalence remains to be determined.
Abstract: A carbapenem-resistant Acinetobacter baumannii strain was isolated in Brazil in 2004 in which no known carbapenemase gene was detected by PCR. Cloning experiments, followed by expression in Escherichia coli , gave an E. coli recombinant strain expressing a novel carbapenem-hydrolyzing class D β-lactamase (CHDL). OXA-143 showed 88% amino acid sequence identity with OXA-40, 63% identity with OXA-23, and 52% identity with OXA-58. It hydrolyzed penicillins, oxacillin, meropenem, and imipenem but not expanded-spectrum cephalosporins. The bla OXA-143 gene was located on a ca. 30-kb plasmid. After transformation into reference strain A. baumannii ATCC 19606, it conferred resistance to carbapenems. Analysis of the genetic environment of bla OXA-143 revealed that it was associated with neither insertion sequences nor integron structures. However, it was bracketed by similar replicase-encoding genes at both ends, suggesting acquisition through a homologous recombination process. This study identified a novel class D β-lactamase involved in carbapenem resistance in A. baumannii . This enzyme is the first member of a novel subgroup of CHDLs whose prevalence remains to be determined.
TL;DR: It is concluded that the dosCP operon encodes two oxygen sensors that cooperate in the controlled production and removal of c-di-GMP.
Abstract: A commonly observed coupling of sensory domains to GGDEF-class diguanylate cyclases and EAL-class phosphodiesterases has long suggested that c-di-GMP synthesizing and degrading enzymes sense environmental signals. Nevertheless, relatively few signal ligands have been identified for these sensors, and even fewer instances of in vitro switching by ligand have been demonstrated. Here we describe an Escherichia coli two-gene operon, dosCP, for control of c-di-GMP by oxygen. In this operon, the gene encoding the oxygen-sensing c-di-GMP phosphodiesterase Ec Dos (here renamed Ec DosP) follows and is translationally coupled to a gene encoding a diguanylate cyclase, here designated DosC. We present the first characterizations of DosC and a detailed study of the ligand-dose response of DosP. Our results show that DosC is a globin-coupled sensor with an apolar but accessible heme pocket that binds oxygen with a K(d) of 20 microM. The response of DosP activation to increasing oxygen concentration is a complex function of its ligand saturation such that over 80% of the activation occurs in solutions that exceed 30% of air saturation (oxygen >75 microM). Finally, we find that DosP and DosC associate into a functional complex. We conclude that the dosCP operon encodes two oxygen sensors that cooperate in the controlled production and removal of c-di-GMP.
TL;DR: It is demonstrated that short sequences of nucleic acids characteristic for bacterial pathogens such as Brucella abortus, Escherichia coli, and Staphylococcus aureus can be detected at 100 pM levels.
TL;DR: The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria.
Abstract: Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1–Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1–Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities.
TL;DR: The recombinant E. coli strains - MG1655, DH5alpha, S17-1, XL1-Blue and BL21 - the DH5 alpha was found to be the best beta-carotene producer and glycerol as the carbon source for beta- carotene production was foundto be superior to glucose, galactose, xylose and maltose.
TL;DR: Results confirm that commensal E. coli strains can provide a barrier to infection and suggest that it may be possible to construct E. Escherichia coli probiotic strains that prevent growth of pathogenic E.coli strains in the intestine.
Abstract: Different Escherichia coli strains generally have the same metabolic capacity for growth on sugars in vitro, but they appear to use different sugars in the streptomycin-treated mouse intestine (Fabich et al., Infect. Immun. 76:1143-1152, 2008). Here, mice were precolonized with any of three human commensal strains (E. coli MG1655, E. coli HS, or E. coli Nissle 1917) and 10 days later were fed 105 CFU of the same strains. While each precolonized strain nearly eliminated its isogenic strain, confirming that colonization resistance can be modeled in mice, each allowed growth of the other commensal strains to higher numbers, consistent with different commensal E. coli strains using different nutrients in the intestine. Mice were also precolonized with any of five commensal E. coli strains for 10 days and then were fed 105 CFU of E. coli EDL933, an O157:H7 pathogen. E. coli Nissle 1917 and E. coli EFC1 limited growth of E. coli EDL933 in the intestine (103 to 104 CFU/gram of feces), whereas E. coli MG1655, E. coli HS, and E. coli EFC2 allowed growth to higher numbers (106 to 107 CFU/gram of feces). Importantly, when E. coli EDL933 was fed to mice previously co-colonized with three E. coli strains (MG1655, HS, and Nissle 1917), it was eliminated from the intestine (<10 CFU/gram of feces). These results confirm that commensal E. coli strains can provide a barrier to infection and suggest that it may be possible to construct E. coli probiotic strains that prevent growth of pathogenic E. coli strains in the intestine.
TL;DR: The genome sequences revealed that a progenitor of REL606 had been misidentified, explaining initially perplexing differences, and more than half of the 3793 proteins of their basic genomes are predicted to be identical, although approximately 310 appear to be functional in either B or K-12 but not in both.
TL;DR: This method demonstrates the utility of a purely stoichiometric network to predict improved Escherichia coli genotypes that more effectively channel carbon flux toward malonyl coenzyme A (CoA) and other cofactors in an effort to generate recombinant strains with enhanced flavonoid production capacity.
Abstract: Identification of genetic targets able to bring about changes to the metabolite profiles of microorganisms continues to be a challenging task. We have independently developed a cipher of evolutionary design (CiED) to identify genetic perturbations, such as gene deletions and other network modifications, that result in optimal phenotypes for the production of end products, such as recombinant natural products. Coupled to an evolutionary search, our method demonstrates the utility of a purely stoichiometric network to predict improved Escherichia coli genotypes that more effectively channel carbon flux toward malonyl coenzyme A (CoA) and other cofactors in an effort to generate recombinant strains with enhanced flavonoid production capacity. The engineered E. coli strains were constructed first by the targeted deletion of native genes predicted by CiED and then second by incorporating selected overexpressions, including those of genes required for the coexpression of the plant-derived flavanones, acetate assimilation, acetyl-CoA carboxylase, and the biosynthesis of coenzyme A. As a result, the specific flavanone production from our optimally engineered strains was increased by over 660% for naringenin (15 to 100 mg/liter/optical density unit [OD]) and by over 420% for eriodictyol (13 to 55 mg/liter/OD).
TL;DR: Investigating whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors found that complement, and in particular FH, not only plays an important role in atypical HUS but most probably also in EH EC-induced HUS.
Abstract: Infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of hemolytic uremic syndrome (HUS). Shiga toxins (Stxs), especially Stx2, are believed to represent major virulence factors of EHEC, contributing to HUS pathogenesis. Beside EHEC-associated HUS, there are hereditary atypical forms of HUS, which are mostly caused by mutations of complement regulators. The aim of the present study was to investigate whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors. Purified Stx2 markedly activated complement via the alternative pathway and was found to bind to factor H (FH), however, only when it was active. No apparent cleavage or destruction of FH was visible, and cofactor activity in fluid phase was unaffected, but clearly delayed for surface-attached FH, where it is essential for host cell protection. Binding studies using FH constructs revealed that Stx2 binds to short consensus repeats (SCRs) 6-8 and SCRs18-20, but not to SCRs16-17, i.e., to regions involved in the surface recognition function of FH. In conclusion, complement, and in particular FH, not only plays an important role in atypical HUS, but most probably also in EHEC-induced HUS.
TL;DR: The transmissible antigen in enteropathogenic E. coli strains from calf and lamb, previously called Kco, is established as the E. bacteria K99 antigen, probably of protein nature since it is destroyed by heating.
Abstract: The transmissible antigen in enteropathogenic E. coli strains from calf and lamb, previously called Kco, is established as the E. coli K99 antigen. It is probably of protein nature since it is destroyed by heating. It is pointed out that other antigens present, growth medium and unknown factors are of great importance for the demonstration of this antigen.
TL;DR: The constructed battery of 19 recombinant luminescent bacterial strains exhibits several novel aspects as it contains metal sensor strains with similar metal- response elements in different host bacteria; metal sensor strain with metal-response elements indifferent copies and a "lights-off" construct (control) for every constructed recombinantMetal sensor strain.
Abstract: Recombinant whole-cell sensors have already proven useful in the assessment of the bioavailability of environmental pollutants like heavy metals and organic compounds In this work 19 recombinant bacterial strains representing various Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas fluorescens) bacteria were constructed to express the luminescence encoding genes luxCDABE (from Photorhabdus luminescens) as a response to bioavailable heavy metals ("lights-on" metal sensors containing metal-response elements, 13 strains) or in a constitutive manner ("lights-off" constructs, 6 strains) The bioluminescence of all 13 "lights-on" metal sensor strains was expressed as a function of the sub-toxic metal concentrations enabling the quantitative determination of metals bioavailable for these strains Five sensor strains, constructed for detecting copper and mercury, proved to be target metal specific, whereas eight other sensor strains were simultaneously induced by Cd2+, Hg2+, Zn2+and Pb2+ The lowest limits of determination of the "lights-on" sensor strains for the metals tested in this study were (μg l-1): 0002 of CH3HgCl, 003 of HgCl2, 18 of CdCl2, 33 of Pb(NO3)2, 1626 of ZnSO4, 24 of CuSO4 and 340 of AgNO3 In general, the sensitivity of the "lights-on" sensor strains was mostly dependent on the metal-response element used while the selection of host bacterium played a relatively minor role In contrast, toxicity of metals to the "lights-off" strains was only dependent on the bacterial host so that Gram-positive strains were remarkably more sensitive than Gram-negative ones The constructed battery of 19 recombinant luminescent bacterial strains exhibits several novel aspects as it contains i) metal sensor strains with similar metal-response elements in different host bacteria; ii) metal sensor strains with metal-response elements in different copies and iii) a "lights-off" construct (control) for every constructed recombinant metal sensor strain To our knowledge, no Gram-positive metal sensor expressing a full bacterial bioluminescence cassette (luxCDABE) has been constructed previously