Showing papers on "Escherichia coli published in 2008"
TL;DR: The results of the present study suggest that silver ions may cause S. aureus and E. coli bacteria to reach an ABNC state and eventually die.
Abstract: The antibacterial effect and mechanism of action of a silver ion solution that was electrically generated were investigated for Staphylococcus aureus and Escherichia coli by analyzing the growth, morphology, and ultrastructure of the bacterial cells following treatment with the silver ion solution. Bacteria were exposed to the silver ion solution for various lengths of time, and the antibacterial effect of the solution was tested using the conventional plate count method and flow cytometric (FC) analysis. Reductions of more than 5 log10 CFU/ml of both S. aureus and E. coli bacteria were confirmed after 90 min of treatment with the silver ion solution. Significant reduction of S. aureus and E. coli cells was also observed by FC analysis; however, the reduction rate determined by FC analysis was less than that determined by the conventional plate count method. These differences may be attributed to the presence of bacteria in an active but nonculturable (ABNC) state after treatment with the silver ion solution. Transmission electron microscopy showed considerable changes in the bacterial cell membranes upon silver ion treatment, which might be the cause or consequence of cell death. In conclusion, the results of the present study suggest that silver ions may cause S. aureus and E. coli bacteria to reach an ABNC state and eventually die.
1,616 citations
TL;DR: A synthetic pathway is engineered in Escherichia coli and the production of 1-butanol is demonstrated from this non-native user-friendly host, showing promise for using E. coli for 1- butanol production.
900 citations
TL;DR: Pangenomic calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors, which should provide the basis for future functional work on this important group of pathogens.
Abstract: Whole-genome sequencing has been skewed toward bacterial pathogens as a consequence of the prioritization of medical and veterinary diseases. However, it is becoming clear that in order to accurately measure genetic variation within and between pathogenic groups, multiple isolates, as well as commensal species, must be sequenced. This study examined the pangenomic content of Escherichia coli. Six distinct E. coli pathovars can be distinguished using molecular or phenotypic markers, but only two of the six pathovars have been subjected to any genome sequencing previously. Thus, this report provides a seminal description of the genomic contents and unique features of three unsequenced pathovars, enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. We also determined the first genome sequence of a human commensal E. coli isolate, E. coli HS, which will undoubtedly provide a new baseline from which workers can examine the evolution of pathogenic E. coli. Comparison of 17 E. coli genomes, 8 of which are new, resulted in identification of ∼2,200 genes conserved in all isolates. We were also able to identify genes that were isolate and pathovar specific. Fewer pathovar-specific genes were identified than anticipated, suggesting that each isolate may have independently developed virulence capabilities. Pangenome calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors. This comparative study of the species E. coli, while descriptive, should provide the basis for future functional work on this important group of pathogens.
806 citations
TL;DR: E. coli ST131 and ST405 and multidrug-resistant IncFII plasmids may determine spread of this lactamase.
Abstract: We analyzed 43 CTX-M-15-producing Escherichia coli isolates and 6 plasmids encoding the blaCTX-M-15 gene from Canada, India, Kuwait, France, Switzerland, Portugal, and Spain. Most isolates belonged to phylogroups B2 (50%) and D (25%). An EC-B2 strain of clonal complex sequence type (ST) 131 was detected in all countries; other B2 isolates corresponded to ST28, ST405, ST354, and ST695 from specific areas. EC-D strains were clonally unrelated but isolates from 3 countries belonged to ST405. All CTX-M-15 plasmids corresponded to IncFII group with overrepresentation of 3 HpaI-digested plasmid DNA profiles (A, B and C; 85-120kb, similarity > or =70%). Plasmid A was detected in EC-B2 strains (ST131, ST354, or ST405), plasmid C was detected in B2 and D strains, and plasmid B was confined to worldwide-disseminated ST131. Most plasmids contained blaOXA-1, aac(6')-Ib-cr, and blaTEM-1. Worldwide dissemination of CTX-M-15 seems to be determined by clonal complexes ST131 and ST405 and multidrug-resistant IncFII plasmids.
739 citations
TL;DR: Spread of mobile genetic elements, mainly epidemic plasmids, and the dispersion of specific clones have been responsible for the increase in ESBL-producing isolates, such as those with TEM-4, Tem-24, TEM
646 citations
TL;DR: The mechanisms by which virulence factors promote UPEC survival and growth within the urinary tract are discussed and Comparisons are made between UPEC and other strains of extraintestinal pathogenic E. coli that are distinct in their abilities to colonize the host and cause disease.
560 citations
TL;DR: This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli.
Abstract: Escherichia coli is a predominant species among facultative anaerobic bacteria of the gastrointestinal tract. Both its frequent community lifestyle and the availability of a wide array of genetic tools contributed to establish E. coli as a relevant model organism for the study of surface colonization. Several key factors, including different extracellular appendages, are implicated in E. coli surface colonization and their expression and activity are finely regulated, both in space and time, to ensure productive events leading to mature biofilm formation. This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli.
503 citations
TL;DR: Findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease, and the ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.
Abstract: Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.
453 citations
TL;DR: A derivative strain of E. coli BL21(DE3) is engineered, termed Lemo21(de3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys), which allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains.
Abstract: A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
438 citations
TL;DR: This work systematically improved the synthesis of 1-propanol and 1-butanol through deregulation of amino-acid biosynthesis and elimination of competing pathways.
408 citations
TL;DR: The BCD activity, which was not detected in E. coli previously, was shown by performing the procedure from cell extract preparation to activity measurement under anaerobic condition and the etfA and etfB co-expression was found to be essential for the B CD activity.
Abstract: A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.
TL;DR: Compared the pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in vitro, it is found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time.
Abstract: The carbon sources that support the growth of pathogenic Escherichia coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coli EDL933 grows primarily as single cells dispersed within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in vitro, using a mixture of the sugars known to be present in cecal mucus, and found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time. We conducted systematic mutation analyses of E. coli EDL933 and E. coli MG1655 by using lesions in the pathways used for catabolism of 13 mucus-derived sugars and five other compounds for which the corresponding bacterial gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic backgrounds was fed to streptomycin-treated mice, together with the respective wild-type parent strain, and their colonization was monitored by fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota.
TL;DR: The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.
Abstract: A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (1010, 109, and 108 PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 109 PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.
TL;DR: The trend is that heterologous expression of some proteins in bacteria can be improved by altering codon preference, but that this effect can be generally recapitulated by introducing rare codon tRNAs into the host cell.
TL;DR: It is demonstrated that human bacteremia strains distribute over the entire span of E. coli phylogenetic diversity and that CCs represent important phylogenetic units for pathogenesis and comparative genomics.
Abstract: Extraintestinal pathogenic Escherichia coli (ExPEC) strains represent a huge public health burden. Knowledge of their clonal diversity and of the association of clones with genomic content and clinical features is a prerequisite to recognize strains with a high invasive potential. In order to provide an unbiased view of the diversity of E. coli strains responsible for bacteremia, we studied 161 consecutive isolates from patients with positive blood culture obtained during one year in two French university hospitals. We collected precise clinical information, multilocus sequence typing (MLST) data and virulence gene content for all isolates. A subset representative of the clonal diversity was subjected to comparative genomic hybridization (CGH) using 2,324 amplicons from the flexible gene pool of E. coli. Recombination-insensitive phylogenetic analysis of MLST data in combination with the ECOR collection revealed that bacteremic E. coli isolates were highly diverse and distributed into five major lineages, corresponding to the classical E. coli phylogroups (A+B1, B2, D and E) and group F, which comprises strains previously assigned to D. Compared to other strains of phylogenetic group B2, strains belonging to MLST-derived clonal complexes (CCs) CC1 and CC4 were associated (P < 0.05) with a urinary origin. In contrast, no CC appeared associated with severe sepsis or unfavorable outcome of the bacteremia. CGH analysis revealed genomic characteristics of the distinct CCs and identified genomic regions associated with CC1 and/or CC4. Our results demonstrate that human bacteremia strains distribute over the entire span of E. coli phylogenetic diversity and that CCs represent important phylogenetic units for pathogenesis and comparative genomics.
TL;DR: The study has revealed that E. coli in raw foods is a significant reservoir of resistance and virulence genes.
TL;DR: The CTX-M-15-producing E. coli diffusing clone is associated with a high level of antibiotic resistance and with high virulence, showing that, under certain selective pressures, the previously observed trade-off between resistance and virulence may not apply.
Abstract: Objectives A clone of CTX-M-15-producing Escherichia coli has recently been reported to be spreading through Europe and Africa. The aim of this work was to thoroughly characterize this clone. Materials and methods Representative isolates of this clone were subjected to multilocus sequence typing, O typing, virulence gene detection, adhesion assay on human cells, biofilm production assay and mouse lethality assay. Results The clone: (i) belongs to a unique B2 phylogenetic subgroup encompassing the pyelonephritogenic diffusely adhering EC7372 strain; (ii) exhibits a specific O25b molecular subtype; (iii) is identical to the E. coli clone O25:H4-ST131 producing CTX-M-15; (iv) produces biofilm; and (v) is highly virulent in mice despite lacking classical extraintestinal pathogenicity islands (except for high pathogenicity island) and the afa/dra gene. Conclusions The CTX-M-15-producing E. coli diffusing clone is associated with a high level of antibiotic resistance and with high virulence, showing that, under certain selective pressures, the previously observed trade-off between resistance and virulence may not apply.
TL;DR: It was shown that the ethanol yields of strains with minimized metabolic functionality closely matched the theoretical predictions, and catabolite repression was completely absent during anaerobic growth, resulting in the simultaneous utilization of pentoses and hexoses for ethanol production.
Abstract: To obtain an efficient ethanologenic Escherichia coli strain, we reduced the functional space of the central metabolic network, with eight gene knockout mutations, from over 15,000 pathway possibilities to 6 pathway options that support cell function. The remaining pathways, identified by elementary mode analysis, consist of four pathways with non-growth-associated conversion of pentoses and hexoses into ethanol at theoretical yields and two pathways with tight coupling of anaerobic cell growth with ethanol formation at high yields. Elimination of three additional genes resulted in a strain that selectively grows only on pentoses, even in the presence of glucose, with a high ethanol yield. We showed that the ethanol yields of strains with minimized metabolic functionality closely matched the theoretical predictions. Remarkably, catabolite repression was completely absent during anaerobic growth, resulting in the simultaneous utilization of pentoses and hexoses for ethanol production.
TL;DR: The data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces, and several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, influenced killing rates.
Abstract: Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.
TL;DR: Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity, and this is the first such group for which structures and DNA sequences have been determined for all O antigens.
Abstract: This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.
TL;DR: Findings show that secreted antimicrobial peptides are retained by the surface-overlaying mucus and thereby provide a combined physical and antibacterial barrier to prevent bacterial attachment and invasion.
Abstract: Objectives: The intestinal mucosa is constantly exposed to a dense and highly dynamic microbial flora and challenged by a variety of enteropathogenic bacteria. Antibacterial protection is provided in part by Paneth cell-derived antibacterial peptides such as the α-defensins. The mechanism of peptide-mediated antibacterial control and its functional importance for gut homeostasis has recently been appreciated in patients with Crohn’s ileitis. In the present study, the spatial distribution of antimicrobial peptides was analysed within the small intestinal anatomical compartments such as the intestinal crypts, the overlaying mucus and the luminal content. Methods: Preparations from the different intestinal locations as well as whole mouse small intestine were extracted and separated by reversed-phase high-performance liquid chromatography. Antibacterial activity was determined in extracts, and the presence of antimicrobial peptides/proteins was confirmed by N-terminal sequencing, mass spectrometry analysis and immunodetection. Results: The secreted antibacterial activity was largely confined to the layer of mucus, whereas only minute amounts of activity were noted in the luminal content. The extractable activity originating from either crypt/mucus/lumen compartments respectively (given as a percentage) was for Listeria monocytogenes , 48 (4)/44 (4)/8 (8); Enterococcus faecalis , 44 (10)/49 (3)/7 (7); Bacterium megaterium , 56 (4)/42 (3)/2 (1); Streptococcus pyogenes , 48 (4)/46 (3)/6 (6); Escherichia coli , 46 (4)/47 (3)/7 (7); and Salmonella enterica sv. Typhimurium, 38 (3)/43 (7)/19 (10). A spectrum of antimicrobial peptides was identified in isolated mucus, which exhibited strong and contact-dependent antibacterial activity against both commensal and pathogenic bacteria. Conclusion: These findings show that secreted antimicrobial peptides are retained by the surface-overlaying mucus and thereby provide a combined physical and antibacterial barrier to prevent bacterial attachment and invasion. This distribution facilitates high local peptide concentration on vulnerable mucosal surfaces, while still allowing the presence of an enteric microbiota.
TL;DR: Covalent modification of beta-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.
Abstract: The linear homopolymer poly-beta-1,6-N-acetyl-D-glucosamine (beta-1,6-GlcNAc; PGA) serves as an adhesin for the maintenance of biofilm structural stability in diverse eubacteria. Its function in Escherichia coli K-12 requires the gene products of the pgaABCD operon, all of which are necessary for biofilm formation. PgaC is an apparent glycosyltransferase that is required for PGA synthesis. Using a monoclonal antibody directed against E. coli PGA, we now demonstrate that PgaD is also needed for PGA formation. The deletion of genes for the predicted outer membrane proteins PgaA and PgaB did not prevent PGA synthesis but did block its export, as shown by the results of immunoelectron microscopy (IEM) and antibody adsorption assays. IEM also revealed a conditional localization of PGA at the cell poles, the initial attachment site for biofilm formation. PgaA contains a predicted beta-barrel porin and a superhelical domain containing tetratricopeptide repeats, which may mediate protein-protein interactions, implying that it forms the outer membrane secretin for PGA. PgaB contains predicted carbohydrate binding and polysaccharide N-deacetylase domains. The overexpression of pgaB increased the primary amine content (glucosamine) of PGA. Site-directed mutations targeting the N-deacetylase catalytic activity of PgaB blocked PGA export and biofilm formation, implying that N-deacetylation promotes PGA export through the PgaA porin. The results of previous studies indicated that N-deacetylation of beta-1,6-GlcNAc in Staphylococcus epidermidis by the PgaB homolog, IcaB, anchors it to the cell surface. The deletion of icaB resulted in release of beta-1,6-GlcNAc into the growth medium. Thus, covalent modification of beta-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.
TL;DR: By comparing five new cyanobactin biosynthetic clusters, an assembly line responsible for the biosynthesis of these diverse peptides is reported, which produced the prenylated antitumor preclinical candidate trunkamide in Escherichia coli culture using genetic engineering.
Abstract: More than 100 cyclic peptides harboring heterocyclized residues are known from marine ascidians, sponges and different genera of cyanobacteria. Here, we report an assembly line responsible for the biosynthesis of these diverse peptides, now called cyanobactins, both in symbiotic and free-living cyanobacteria. By comparing five new cyanobactin biosynthetic clusters, we produced the prenylated antitumor preclinical candidate trunkamide in Escherichia coli culture using genetic engineering.
TL;DR: The results indicate that HMG-CoA inhibits fatty acid biosynthesis in the microbial host, leading to generalized membrane stress, demonstrating the utility of using transcriptomic and metabolomic methods to optimize synthetic biological systems.
Abstract: Producing complex chemicals using synthetic metabolic pathways in microbial hosts can have many advantages over chemical synthesis but is often complicated by deleterious interactions between pathway intermediates and the host cell metabolism. With the maturation of functional genomic analysis, it is now technically feasible to identify modes of toxicity associated with the accumulation of foreign molecules in the engineered bacterium. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway (V. J. J. Martin et al., Nat. Biotechnol. 21:796-802, 2003). The engineered E. coli strain produced high levels of isoprenoids, but further optimization led to an imbalance in carbon flux and the accumulation of the pathway intermediate 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), which proved to be cytotoxic to E. coli. Using both DNA microarray analysis and targeted metabolite profiling, we have studied E. coli strains inhibited by the intracellular accumulation of HMG-CoA. Our results indicate that HMG-CoA inhibits fatty acid biosynthesis in the microbial host, leading to generalized membrane stress. The cytotoxic effects of HMG-CoA accumulation can be counteracted by the addition of palmitic acid (16:0) and, to a lesser extent, oleic acid (cis-Δ9-18:1) in the growth medium. This work demonstrates the utility of using transcriptomic and metabolomic methods to optimize synthetic biological systems.
TL;DR: A highly sensitive and specific LAMP assay for Escherichia coli that does not require DNA extraction and can detect as few as 10 copies is developed.
Abstract: We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.
TL;DR: E. coli strains capable of high-level flavonoid synthesis through traditional metabolic engineering techniques are developed and demonstrated the efficient and scalable production of plant flavonoids from E. coli for pharmaceutical and nutraceutical applications.
Abstract: Plant flavonoid polyphenols continue to find increasing pharmaceutical and nutraceutical applications; however their isolation, especially of pure compounds, from plant material remains an underlying challenge. In the past Escherichia coli, one of the most well-characterized microorganisms, has been utilized as a recombinant host for protein expression and heterologous biosynthesis of small molecules. However, in many cases the expressed protein activities and biosynthetic efficiency are greatly limited by the host cellular properties, such as precursor and cofactor availability and protein or product tolerance. In the present work, we developed E. coli strains capable of high-level flavonoid synthesis through traditional metabolic engineering techniques. In addition to grafting the plant biosynthetic pathways, the methods included engineering of an alternative carbon assimilation pathway and the inhibition of competitive reaction pathways in order to increase intracellular flavonoid backbone precursors a...
TL;DR: The essential inner-membrane protein MviN (renamed MurJ) is identified as a likely candidate for the peptidoglycan flippase in Escherichia coli and genetic and biochemical data are presented that confirm the requirement of MurJ for peptidlycercan biosynthesis and that are in agreement with a role ofMurJ as a flippases.
Abstract: Peptidoglycan is a cell-wall glycopeptide polymer that protects bacteria from osmotic lysis. Whereas in Gram-positive bacteria it also serves as scaffold for many virulence factors, in Gram-negative bacteria, peptidoglycan is an anchor for the outer membrane. For years, we have known the enzymes required for the biosynthesis of peptidoglycan; what was missing was the flippase that translocates the lipid-anchored precursors across the cytoplasmic membrane before their polymerization into mature peptidoglycan. Using a reductionist bioinformatics approach, I have identified the essential inner-membrane protein MviN (renamed MurJ) as a likely candidate for the peptidoglycan flippase in Escherichia coli. Here, I present genetic and biochemical data that confirm the requirement of MurJ for peptidoglycan biosynthesis and that are in agreement with a role of MurJ as a flippase. Because of its essential nature, MurJ could serve as a target in the continuing search for antimicrobial compounds.
TL;DR: Identification of plasmid-mediated qnr genes outside Enterobacteriaceae underlines a possible diffusion of those resistance determinants within gram-negative rods.
Abstract: We searched for plasmid-mediated quinolone resistance determinants of the Qnr type in several water samples collected at diverse locations from the Seine River (Paris, France). The qnrS2 genes were identified from Aeromonas punctata subsp. punctata and A. media. The qnrS2 gene was located on IncU-type plasmids in both isolates, which resulted in increased MIC values of quinolones and fluoroquinolones, once they were transferred into Escherichia coli. The qnrS2 gene identified in A. punctata was part of novel genetic structure corresponding to a mobile insertion cassette element. This identification of plasmid-mediated qnr genes outside Enterobacteriaceae underlines a possible diffusion of those resistance determinants within gram-negative rods.
TL;DR: The bioinformatic and experimental results suggest that many bacteria may not generally oxidatively fold proteins, and implicate the bacterial homolog of the enzyme vitamin K epoxide reductase, a protein required for blood clotting in humans, as part of a disulfide bond formation pathway present in several major bacterial phyla.
Abstract: Protein disulfide bond formation contributes to the folding and activity of many exported proteins in bacteria. However, information about disulfide bond formation is limited to only a few bacterial species. We used a multifaceted bioinformatic approach to assess the capacity for disulfide bond formation across this biologically diverse group of organisms. We combined data from a cysteine counting method, in which a significant bias for even numbers of cysteine in proteins is taken as an indicator of disulfide bond formation, with data on the presence of homologs of known disulfide bond formation enzymes. These combined data enabled us to make predictions about disulfide bond formation in the cell envelope across bacterial species. Our bioinformatic and experimental results suggest that many bacteria may not generally oxidatively fold proteins, and implicate the bacterial homolog of the enzyme vitamin K epoxide reductase, a protein required for blood clotting in humans, as part of a disulfide bond formation pathway present in several major bacterial phyla.