scholarly journals Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Philipp Keller ◽  
Elad Noor ◽  
Fabian Meyer ◽  
Michael A. Reiter ◽  
Stanislav Anastassov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Triosephosphate Isomerase ◽  
Methanol Conversion ◽  
Adaptive Laboratory Evolution ◽  
Fructose 1,6 Bisphosphatase ◽  
Food And Feed ◽  
Balance Analysis ◽  
Ribulose Monophosphate Cycle ◽  
High Degree ◽  
Rump Cycle

Abstract Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Although progress has been made towards establishing Escherichia coli as a platform organism for methanol conversion via the energy efficient ribulose monophosphate (RuMP) cycle, engineering strains that rely solely on methanol as a carbon source remains challenging. Here, we apply flux balance analysis to comprehensively identify methanol-dependent strains with high potential for adaptive laboratory evolution. We further investigate two out of 1200 candidate strains, one with a deletion of fructose-1,6-bisphosphatase (fbp) and another with triosephosphate isomerase (tpiA) deleted. In contrast to previous reported methanol-dependent strains, both feature a complete RuMP cycle and incorporate methanol to a high degree, with up to 31 and 99% fractional incorporation into RuMP cycle metabolites. These strains represent ideal starting points for evolution towards a fully methylotrophic lifestyle.

scholarly journals Fermentative Utilization of Glycerol by Escherichia coli and Its Implications for the Production of Fuels and Chemicals

2007 ◽  
Vol 74 (4) ◽  
pp. 1124-1135 ◽  
Author(s):  
Abhishek Murarka ◽  
Yandi Dharmadi ◽  
Syed Shams Yazdani ◽  
Ramon Gonzalez
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Fermentation ◽  
Glycerol Metabolism ◽  
Nuclear Magnetic Resonance Analysis ◽  
Glycerol Fermentation ◽  
Glycerol Utilization ◽  
Negative Effect ◽  
Balance Analysis ◽  
Fuels And Chemicals ◽  
High Degree

ABSTRACT Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ± 0.003 h−1. The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-13C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-13C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (∼95%) and redox (∼96%) balances. On the other hand, cultivation conditions that prevent H2 accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H2 is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.

scholarly journals The orientation of iron–sulphur clusters in membrane multilayers prepared from aerobically-grown Escherichia coli K12 and a cytochrome-deficient mutant

1980 ◽  
Vol 190 (2) ◽  
pp. 385-393 ◽  
Author(s):  
Haywood Blum ◽  
Robert K. Poole ◽  
Tomoko Ohnishi
Keyword(s):  
Magnetic Field ◽  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Deficient Mutant ◽  
E Coli ◽  
Membrane Particles ◽  
Degree Of Orientation ◽  
High Degree ◽  
Cytochrome Content ◽  
The Magnetic Field

1. Membrane particles prepared from ultrasonically-disrupted, aerobically-grown Escherichia coli were centrifuged on to a plastic film that was supported perpendicular to the centrifugal field to yield oriented membrane multilayers. In such preparations, there is a high degree of orientation of the planes of the membranes such that they lie parallel to each other and to the supporting film. 2. When dithionite- or succinate-reduced multilayers are rotated in the magnetic field of an e.p.r. spectrometer, about an axis lying in the membrane plane, angular-dependent signals from an iron–sulphur cluster at gx=1.92, gy=1.93 and gz=2.02 are seen. The g=1.93 signal has maximal amplitude when the plane of the multilayer is perpendicular to the magnetic field. Conversely, the g=2.02 signal is maximal when the plane of the multilayer is parallel with the magnetic field. 3. Computer simulations of the experimental data show that the cluster lies in the cytoplasmic membrane with the gy axis perpendicular to the membrane plane and with the gx and gz axes lying in the membrane plane. 4. In partially-oxidized multilayers, a signal resembling the mitochondrial high-potential iron–sulphur protein (Hipip) is seen whose gz=2.02 axis may be deduced as lying perpendicular to the membrane plane. 5. Appropriate choice of sample temperature and receiver gain reveals two further signals in partially-reduced multilayers: a g=2.09 signal arises from a cluster with its gz axis in the membrane plane, whereas a g=2.04 signal is from a cluster with the gz axis lying along the membrane normal. 6. Membrane particles from a glucose-grown, haem-deficient mutant contain dramatically-lowered levels of cytochromes and exhibit, in addition to the iron–sulphur clusters seen in the parental strain, a major signal at g=1.90. 7. Only the latter may be demonstrated to be oriented in multilayer preparations from the mutant. 8. Comparisons are drawn between the orientations of the iron–sulphur proteins in the cytoplasmic membrane of E. coli and those in mitochondrial membranes. The effects of diminished cytochrome content on the properties of the iron–sulphur proteins are discussed.

scholarly journals Sulfated K5 Escherichia coli Polysaccharide Derivatives as Wide-Range Inhibitors of Genital Types of Human Papillomavirus

2008 ◽  
Vol 52 (4) ◽  
pp. 1374-1381 ◽  
Author(s):  
David Lembo ◽  
Manuela Donalisio ◽  
Marco Rusnati ◽  
Antonella Bugatti ◽  
Maura Cornaglia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Capsular Polysaccharide ◽  
Human Papillomaviruses ◽  
Hpv 16 ◽  
Biological Assays ◽  
Sexually Transmitted ◽  
Wide Range ◽  
Topical Microbicides ◽  
Polysaccharide Derivatives ◽  
High Degree

ABSTRACT Genital human papillomaviruses (HPV) represent the most common sexually transmitted agents and are classified into low or high risk by their propensity to cause genital warts or cervical cancer, respectively. Topical microbicides against HPV may be a useful adjunct to the newly licensed HPV vaccine. A main objective in the development of novel microbicides is to block HPV entry into epithelial cells through cell surface heparan sulfate proteoglycans. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was integrated with innovative biochemical and biological assays to prepare a collection of sulfated K5 derivatives with a backbone structure resembling the heparin/heparan biosynthetic precursor and to test them for their anti-HPV activity. Surface plasmon resonance assays revealed that O-sulfated K5 with a high degree of sulfation [K5-OS(H)] and N,O-sulfated K5 with a high [K5-N,OS(H)] or low [K5-N,OS(L)] sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low levels of sulfation, prevented the interaction between HPV-16 pseudovirions and immobilized heparin. In cell-based assays, K5-OS(H), K5-N,OS(H), and K5-N,OS(L) inhibited HPV-16, HPV-18, and HPV-6 pseudovirion infection. Their 50% inhibitory concentration was between 0.1 and 0.9 μg/ml, without evidence of cytotoxicity. These findings provide insights into the design of novel, safe, and broad-spectrum microbicides against genital HPV infections.

scholarly journals Autotransporters of Escherichia coli: a sequence-based characterization

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2459-2469 ◽  
Author(s):  
Timothy J. Wells ◽  
Makrina Totsika ◽  
Mark A. Schembri
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Domain Architecture ◽  
Bioinformatic Analysis ◽  
Tissue Tropism ◽  
Genome Sequences ◽  
E Coli ◽  
Disease Outcomes ◽  
High Degree ◽  
Encoding Genes

Autotransporter (AT) proteins are found in all Escherichia coli pathotypes and are often associated with virulence. In this study we took advantage of the large number of available E. coli genome sequences to perform an in-depth bioinformatic analysis of AT-encoding genes. Twenty-eight E. coli genome sequences were probed using an iterative approach, which revealed a total of 215 AT-encoding sequences that represented three major groups of distinct domain architecture: (i) serine protease AT proteins, (ii) trimeric AT adhesins and (iii) AIDA-I-type AT proteins. A number of subgroups were identified within each broad category, and most subgroups contained at least one characterized AT protein; however, seven subgroups contained no previously described proteins. The AIDA-I-type AT proteins represented the largest and most diverse group, with up to 16 subgroups identified from sequence-based comparisons. Nine of the AIDA-I-type AT protein subgroups contained at least one protein that possessed functional properties associated with aggregation and/or biofilm formation, suggesting a high degree of redundancy for this phenotype. The Ag43, YfaL/EhaC, EhaB/UpaC and UpaG subgroups were found in nearly all E. coli strains. Among the remaining subgroups, there was a tendency for AT proteins to be associated with individual E. coli pathotypes, suggesting that they contribute to tissue tropism or symptoms specific to different disease outcomes.

scholarly journals Limited Genetic Diversity of blaCMY-2-Containing IncI1-pST12 Plasmids from Enterobacteriaceae of Human and Broiler Chicken Origin in The Netherlands

2020 ◽  
Vol 8 (11) ◽  
pp. 1755
Author(s):  
Evert Drijver ◽  
Joep Stohr ◽  
Jaco Verweij ◽  
Carlo Verhulst ◽  
Francisca Velkers ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Data ◽  
Sequence Similarity ◽  
Sequencing Analysis ◽  
Nucleotide Polymorphisms ◽  
Pan Genome ◽  
Next Generation Sequencing Analysis ◽  
Long Read ◽  
Short Read Sequence ◽  
High Degree

Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1–pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a blaCMY-2 gene and an IncI1–pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine Escherichia coli sequences of broiler origin, four Escherichia coli sequences, and one Salmonella enterica sequence of human origin were selected for the current analysis. A circular contig with the IncI1–pST12 replicon and blaCMY-2 gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1–pST12 plasmids revealed a low number of SNP differences (range of 0–9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of blaCMY-2-containing IncI1–pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.

scholarly journals Virulence Characteristics and Antibiotic Resistance Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Diverse Sources

Antibiotics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 587
Author(s):  
Momna Rubab ◽  
Deog-Hwan Oh
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Shiga Toxin ◽  
Multidrug Resistant ◽  
Control Measures ◽  
Diffusion Method ◽  
Shiga Toxins ◽  
E Coli ◽  
And Control ◽  
High Degree

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen that causes several gastrointestinal ailments in humans across the world. STEC’s ability to cause ailment is attributed to the presence of a broad range of known and putative virulence factors (VFs) including those that encode Shiga toxins. A total of 51 E. coli strains belonging to serogroups O26, O45, O103, O104, O113, O121, O145, and O157 were tested for the presence of nine VFs via PCR and for their susceptibility to 17 frequently used antibiotics using the disc diffusion method. The isolates belonged to eight different serotypes, including eight O serogroups and 12 H types. The frequency of the presence of key VFs were stx1 (76.47%), stx2 (86.27%), eae (100%), ehxA (98.03%), nleA (100%), ureC (94.11%), iha (96.07%), subA (9.80%), and saa (94.11%) in the E. coli strains. All E. coli strains carried seven or more distinct VFs and, among these, four isolates harbored all tested VFs. In addition, all E. coli strains had a high degree of antibiotic resistance and were multidrug resistant (MDR). These results show a high incidence frequency of VFs and heterogeneity of VFs and MDR profiles of E. coli strains. Moreover, half of the E. coli isolates (74.5%) were resistant to > 9 classes of antibiotics (more than 50% of the tested antibiotics). Thus, our findings highlight the importance of appropriate epidemiological and microbiological surveillance and control measures to prevent STEC disease in humans worldwide.

Location of a gene specifying phosphopyruvate synthase activity on the genome of Escherichia coli , K12

1967 ◽  
Vol 168 (1012) ◽  
pp. 281-292 ◽  
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli K12 ◽  
High Degree

A gene which specifies the activity of phosphopyruvate synthase has been located on the genome of Escherichia coli . By interrupted mating of a variety of genetic donor (Hfr) and recipient (F - ) strains of Escherichia coli , K12, which carried alleles of the phosphopyruvate synthase ( pps ) gene, it was established that the pps gene is close to the aro - D marker. This was confirmed by phage-mediated transduction of these genes, which were shown to exhibit a high degree of linkage.

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