scholarly journals Increased Furfural Tolerance Due to Overexpression of NADH-Dependent Oxidoreductase FucO in Escherichia coli Strains Engineered for the Production of Ethanol and Lactate

2011 ◽  
Vol 77 (15) ◽  
pp. 5132-5140 ◽  
Author(s):  
X. Wang ◽  
E. N. Miller ◽  
L. P. Yomano ◽  
X. Zhang ◽  
K. T. Shanmugam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Detoxification Enzymes ◽  
Genetic Traits ◽  
New Approach ◽  
Content Type ◽  
Furfural Tolerance ◽  
Primary Products ◽  
Inhibitory Compounds ◽  
Fermentation Inhibitor ◽  
Chromosomal Mutations

ABSTRACTFurfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (lowKmfor NADPH), is proposed to inhibit the growth and fermentation of xylose inEscherichia coliby competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations inyqhDexpression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression offucOfrom plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms.

scholarly journals Increase in Furfural Tolerance in Ethanologenic Escherichia coli LY180 by Plasmid-Based Expression ofthyA

2012 ◽  
Vol 78 (12) ◽  
pp. 4346-4352 ◽  
Author(s):  
Huabao Zheng ◽  
Xuan Wang ◽  
Lorraine P. Yomano ◽  
Keelnatham T. Shanmugam ◽  
Lonnie O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cellular Response ◽  
De Novo ◽  
Content Type ◽  
Genomic Libraries ◽  
Methyl Donor ◽  
E Coli ◽  
Furfural Tolerance ◽  
Small Benefit ◽  
Ethanologenic Escherichia Coli

ABSTRACTFurfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilisYB886,Escherichia coliNC3, andZymomonas mobilisCP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing thethyAgene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in thede novopathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression ofthyAwas no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA inE. coliand to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA inE. coliwould be expected to increase the cellular requirement for dTMP. Increased expression ofthyA(E. coli,B. subtilis, orZ. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

scholarly journals Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

2014 ◽  
Vol 80 (19) ◽  
pp. 5955-5964 ◽  
Author(s):  
Ryan D. Geddes ◽  
Xuan Wang ◽  
Lorraine P. Yomano ◽  
Elliot N. Miller ◽  
Huabao Zheng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Xylose Fermentation ◽  
Coli Strain ◽  
Mineral Salts ◽  
Genetic Changes ◽  
Content Type ◽  
Furfural Tolerance ◽  
Polyamine Transporter ◽  
Ethanologenic Escherichia Coli ◽  
Time Required

ABSTRACTExpression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenicEscherichia coliLY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression ofyqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion ofpotEin EMFR9 lowered furfural tolerance to that of the parent. Deletion ofpotEandpuuPin LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.

scholarly journals Improving Escherichia coli FucO for Furfural Tolerance by Saturation Mutagenesis of Individual Amino Acid Positions

2013 ◽  
Vol 79 (10) ◽  
pp. 3202-3208 ◽  
Author(s):  
Huabao Zheng ◽  
Xuan Wang ◽  
Lorraine P. Yomano ◽  
Ryan D. Geddes ◽  
Keelnatham T. Shanmugam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Gene ◽  
Free Energy Calculations ◽  
Saturation Mutagenesis ◽  
Interfacial Region ◽  
Wild Type ◽  
Gene Encoding ◽  
Translational Initiation ◽  
Content Type ◽  
Furfural Tolerance

ABSTRACTFurfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. InEscherichia coli, furfural tolerance can be increased by expressing the nativefucOgene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form offucOthat confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-typefucOgene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutantfucOgenes differed by less than 2-fold. TheKm(furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides −7 to +37) were weak for the native gene (−4.1 kcal mol−1) but weaker still for thefucOmutant (−1.0 to −0.1 kcal mol−1). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels.

scholarly journals Urinary Tract Conditions Affect Fosfomycin Activity against Escherichia coli Strains Harboring Chromosomal Mutations Involved in Fosfomycin Uptake

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
G. Martín-Gutiérrez ◽  
F. Docobo-Pérez ◽  
J. Rodriguez-Beltrán ◽  
J. M. Rodríguez-Martínez ◽  
J. Aznar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Practical Interest ◽  
Resistance Mutations ◽  
Urine Ph ◽  
Content Type ◽  
E Coli ◽  
Ph Values ◽  
Tract Infections ◽  
Chromosomal Mutations

ABSTRACTThe steps by whichEscherichia colistrains harboring mutations related to fosfomycin (FOS) resistance arise and spread during urinary tract infections (UTIs) are far from being understood. The aim of this study was to evaluate the effects of urine, pH, and anaerobiosis on FOS activity against a set of isogenic strains carrying the most prevalent chromosomal mutations conferring FOS resistance (ΔuhpT, ΔglpT, ΔcyaA, and ΔptsI), either singly or in combination. We also studied fosfomycin-resistantE. coliclinical isolates from patients with UTI. Our results demonstrate that urinary tract physiological conditions might have a profound impact on FOS activity against strains with chromosomal FOS resistance mutations. Specifically, acidic pH values and anaerobiosis convert most of the strains categorized as resistant to fosfomycin according to the international guidelines to a susceptible status. Therefore, urinary pH values may have practical interest in the management of UTIs. Finally, our results, together with the high fitness cost associated with FOS resistance mutations, might explain the low prevalence of fosfomycin-resistantE. colivariants in UTIs.

scholarly journals Identification of Fecal Input Sites in Spring Water by Selection and Genotyping of Multiresistant Escherichia coli

2011 ◽  
Vol 77 (23) ◽  
pp. 8427-8433 ◽  
Author(s):  
Melanie Wicki ◽  
Fatma Karabulut ◽  
Adrian Auckenthaler ◽  
Richard Felleisen ◽  
Marcel Tanner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Water Quality Management ◽  
Spring Water ◽  
Source Tracking ◽  
Molecular Fingerprinting ◽  
New Approach ◽  
Content Type ◽  
Multiresistant Bacteria ◽  
Selective Agar

ABSTRACTThe localization of fecal input sites is important for water quality management. For this purpose, we have developed a new approach based on a three-step procedure, including a preparatory phase, the screening of multiresistant bacteria using selective agar plates, and a typing phase where selectedEscherichia coliisolates are characterized by antibiotic resistance profiles and molecular fingerprinting techniques (pulsed-field gel electrophoresis [PFGE]). These two well-known source tracking methods were combined in order to reduce cost and effort. This approach was successfully applied under field conditions in a study area located in the north-western part of Switzerland.E. coliisolates from spring water and surface water samples collected in this area were screened with selective agar plates. In this way, 21 different groups, each consisting of strains with the same pattern of antibiotic resistance, were found. Of these, four groups were further analyzed using PFGE. Strains with identical PFGE profiles were detected repeatedly, demonstrating the suitability of this method for the localization of fecal input sites over an extended period of time. Identical PFGE patterns of strains detected in water from two different springs were also found in the stream flowing through the study area. These results demonstrated the applicability of the new approach for the examination of incidents of fecal contamination in drinking water. The advantages of the described approach over genotyping methods currently being used to identify sources of fecal contaminants are a reduction in time, costs, and the effort required. Identical isolates could be identified without the construction of large libraries.

scholarly journals F4- and F18-Positive Enterotoxigenic Escherichia coli Isolates from Diarrhea of Postweaning Pigs: Genomic Characterization

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Vanesa García ◽  
Michela Gambino ◽  
Karl Pedersen ◽  
Svend Haugegaard ◽  
John Elmerdahl Olsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Virulence Genes ◽  
The United States ◽  
Enterotoxigenic Escherichia Coli ◽  
Genetic Traits ◽  
Content Type ◽  
Pig Production ◽  
Clonal Group ◽  
The Impact

ABSTRACT This study aimed to characterize in silico enterotoxigenic Escherichia coli F4- and F18-positive isolates (n = 90) causing swine postweaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance, prophage content, and metal tolerance rates. F4 strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 sequence types (STs). F18 strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23, and ST42. The highest rates of antimicrobial resistance were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and ampicillin. No resistance was found toward ciprofloxacin, cefotaxime, ceftiofur, and colistin. Genes conferring tolerance to copper (showing the highest diversity), cadmium, silver, and zinc were predicted in all genomes. Enterotoxin genes (ltcA, 100% F4, 62% F18; astA, 100% F4, 38.1% F18; sta, 18.8% F4, 38.1% F18; stb, 100% F4, 76.2% F18) and fimbria-encoding genes typed as F4ac and F18ac were detected in all strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. A highly diverse variety of plasmids was found; IncFII, IncFIB, and IncFIC were prevalent among F4 isolates, while IncI1 and IncX1 were dominant among F18 strains. Interestingly, F4 isolates from the early 1990s belonged to the same clonal group detected for most of the F4 strains from 2018 to 2019 (ONT:H10-A-ST100-CH27-0). The small number of single-nucleotide polymorphism differences between the oldest and recent F4 ST100 isolates suggests a relatively stable genome. Overall, the isolates analyzed in this study showed remarkably different genetic traits depending on the fimbria type. IMPORTANCE Diarrhea in the postweaning period due to enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by zinc oxide administration (to be discontinued by 2022). In addition, a breeding program has been implemented that aims to reduce the prevalence of this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among diarrhea-associated isolates by whole-genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC ST100 isolates from China, the United States, and Spain. High rates of AMR and high diversity of virulence genes were detected. The characterization of diarrhea-related ETEC is important for understanding the disease epidemiology and pathogenesis and for implementation of new strategies aiming to reduce the impact of the disease in pig production.

scholarly journals Setting the Stage: Genes Controlling Mechanosensation and Ca2+ Signaling in Escherichia coli

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
R. Gary Sawers
Keyword(s):  
Escherichia Coli ◽  
Proton Motive Force ◽  
Current Evidence ◽  
Membrane Biogenesis ◽  
New Approach ◽  
Content Type ◽  
Gene Knockouts ◽  
Keio Collection ◽  
Outer Membrane Biogenesis

ABSTRACT Although mechanistic understanding of calcium signaling in bacteria remains inchoate, current evidence clearly links Ca2+ signaling with membrane potential and mechanosensation. Adopting a radically new approach, Luder et al. scanned the Keio collection of Escherichia coli gene knockouts (R. Luder, G. N. Bruni, and J. M. Kralj, J Bacteriol 203:e00509-20, 2021, https://doi.org/10.1128/JB.00509-20) to identify mutations that cause changes in Ca2+ transients. They identify genes associating Ca2+ signaling with outer membrane biogenesis, proton motive force, and, surprisingly, long-term DNA damage. Their work has major implications for electrophysiological communication between bacteria and their environment.

scholarly journals Detection of Escherichia coli O104 in the Feces of Feedlot Cattle by a Multiplex PCR Assay Designed To Target Major Genetic Traits of the Virulent Hybrid Strain Responsible for the 2011 German Outbreak

2013 ◽  
Vol 79 (11) ◽  
pp. 3522-3525 ◽  
Author(s):  
Z. D. Paddock ◽  
J. Bai ◽  
X. Shi ◽  
D. G. Renter ◽  
T. G. Nagaraja
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Feedlot Cattle ◽  
Pcr Assay ◽  
Hybrid Strain ◽  
Genetic Traits ◽  
Fecal Samples ◽  
Content Type ◽  
E Coli ◽  
Multiplex Pcr Assay

ABSTRACTA multiplex PCR was designed to detectEscherichia coliO104:H4, a hybrid pathotype of Shiga toxigenic and enteroaggregativeE. coli, in cattle feces. A total of 248 fecal samples were tested, and 20.6% were positive for serogroup O104. The O104 isolates did not carry genes characteristic of the virulent hybrid strain.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Draft Genome Sequences of Escherichia coli Strains FP2 and FP3, Isolated from the Canada Goose (Branta canadensis)

2018 ◽  
Vol 7 (9) ◽  
Author(s):  
Allison L. Denny ◽  
Susan E. Arruda
Keyword(s):  
Escherichia Coli ◽  
Draft Genome ◽  
Branta Canadensis ◽  
Canada Goose ◽  
Genome Sequences ◽  
Content Type

Draft genomes of two strains of Escherichia coli, FP2 and FP3, isolated from the feces of the Canada goose (Branta canadensis), were sequenced. Genome sizes were 5.26 Mb with a predicted G+C content of 50.54% (FP2) and 5.07 Mb with a predicted G+C content of 50.41% (FP3).

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