scholarly journals Regeneration ofEscherichia colifrom Minicells through Lateral Gene Transfer

2018 ◽  
Vol 200 (9) ◽  
Author(s):  
Hideki Kobayashi
Keyword(s):  
Genomic Dna ◽  
Living System ◽  
Biologically Active ◽  
Pulse Field ◽  
Natural Behavior ◽  
Content Type ◽  
E Coli ◽  
Pulse Field Gel Electrophoresis ◽  
The Difference ◽  
Genome Donors

ABSTRACTRecently, artificial life has been created with artificial materials and methods. Life can be created when genomic DNA molecules are integrated in liposomes containing biochemical reactions for biogenic needs. However, it is not yet known whether the integration of these parts will be able to occur in nature and constitute a living system. I planned to regenerate bacteria from biologically active liposomes by inserting genomic DNA using only natural materials and methods. Minicells ofEscherichia coli, containing plasmids and activated SOS proteins, act as protocells. Four newE. colistrains were regenerated from minicells by inserting the genomes by using the system for conjugation between F−and Hfr strains. Cells of the four regenerated strains showed the same genetic markers as the two genome donors. Pulse-field gel electrophoresis of their genomes showed admixing of those of both donors. In addition, the genomes of the four regenerated strains had chimeric genome of the two donors. These results show that synthesis of life can occur in nature without artificial arrangement.IMPORTANCEWhat is the difference between inanimate objects and organisms? Organisms always have genomic DNA. When organisms lose their genomes, they can neither grow nor reproduce. As the result, organisms turn into inanimate objects without their genomes. In this study, I regenerated microbes from cells that had lost their genomes (cell corpses) by inserting another genome. All steps of regeneration used the natural behavior of microbes. The same regeneration of microbes could happen in nature. These primitive lives have plasticity, which accelerates evolution and provides various kinds of life in the world.

scholarly journals Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Shota Nagamine ◽  
Chengwei Liu ◽  
Jumpei Nishishita ◽  
Takuto Kozaki ◽  
Kaho Sogahata ◽  
...  
Keyword(s):  
Natural Products ◽  
Aspergillus Oryzae ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Genome Mining ◽  
Biologically Active ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Content System ◽  
Basidiomycete Fungi

ABSTRACT Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining. IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

scholarly journals Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

2014 ◽  
Vol 80 (7) ◽  
pp. 2216-2228 ◽  
Author(s):  
Rebekah A. Frampton ◽  
Corinda Taylor ◽  
Angela V. Holguín Moreno ◽  
Sandra B. Visnovsky ◽  
Nicola K. Petty ◽  
...  
Keyword(s):  
Host Range ◽  
Pseudomonas Syringae ◽  
Management Strategies ◽  
Pulse Field ◽  
Resistant Bacteria ◽  
Bacterial Canker ◽  
Content Type ◽  
Narrow Host Range ◽  
Pulse Field Gel Electrophoresis ◽  
Isolation And Characterization

ABSTRACTPseudomonas syringaepv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidiasp.). Since 2008, a global outbreak ofP. syringaepv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance inP. syringaepv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active againstP. syringaepv. actinidiae. Extensive host range testing onP. syringaepv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to theCaudoviralesand were analyzed based on morphology and genome size, which showed them to be representatives ofMyoviridae,Podoviridae, andSiphoviridae. Twenty-oneMyoviridaemembers have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of theseMyoviridaemembers were sequenced, and each was unique. The most closely related sequenced phages were a group infectingPseudomonas aeruginosaand characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization ofP. syringaepv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.

scholarly journals Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression

2012 ◽  
Vol 78 (10) ◽  
pp. 3622-3629 ◽  
Author(s):  
Matthew D. McMahon ◽  
Changhui Guan ◽  
Jo Handelsman ◽  
Michael G. Thomas
Keyword(s):  
Functional Expression ◽  
Streptomyces Lividans ◽  
Biologically Active ◽  
Functional Screening ◽  
Metagenomic Dna ◽  
Content Type ◽  
E Coli ◽  
Metagenomic Libraries ◽  
Pooled Dna ◽  
Surrogate Host

ABSTRACTMost functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA inEscherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries inStreptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries inS. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native toS. lividans. All bioactive clones were functional inS. lividansbut not inE. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

scholarly journals Molecular Characterization of Fluoroquinolone Resistance in Nontypeable Haemophilus influenzae Clinical Isolates

2014 ◽  
Vol 59 (1) ◽  
pp. 461-466 ◽  
Author(s):  
Carmen Puig ◽  
José Manuel Tirado-Vélez ◽  
Laura Calatayud ◽  
Fe Tubau ◽  
Junkal Garmendia ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Nalidixic Acid ◽  
Respiratory Infections ◽  
Pulse Field ◽  
Fluoroquinolone Resistance ◽  
Chronic Obstructive ◽  
Nontypeable Haemophilus Influenzae ◽  
High Genetic Diversity ◽  
Content Type ◽  
Pulse Field Gel Electrophoresis

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a common cause of respiratory infections in adults, who are frequently treated with fluoroquinolones. The aims of this study were to characterize the genotypes of fluoroquinolone-resistant NTHi isolates and their mechanisms of resistance. Among 7,267H. influenzaeisolates collected from adult patients from 2000 to 2013, 28 (0.39%) were ciprofloxacin resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria. In addition, a nalidixic acid screening during 2010 to 2013 detected five (0.23%) isolates that were ciprofloxacin susceptible but nalidixic acid resistant. Sequencing of their quinolone resistance-determining regions and genotyping by pulse-field gel electrophoresis and multilocus sequence typing of the 25 ciprofloxacin-resistant isolates available and all 5 nalidixic acid-resistant isolates were performed. In the NTHi isolates studied, two mutations producing changes in two GyrA residues (Ser84, Asp88) and/or two ParC residues (Ser84, Glu88) were associated with increased fluoroquinolone MICs. Strains with one or two mutations (n= 15) had ciprofloxacin and levofloxacin MICs of 0.12 to 2 μg/ml, while those with three or more mutations (n= 15) had MICs of 4 to 16 μg/ml. Long persistence of fluoroquinolone-resistant strains was observed in three chronic obstructive pulmonary disease patients. High genetic diversity was observed among fluoroquinolone-resistant NTHi isolates. Although fluoroquinolones are commonly used to treat respiratory infections, the proportion of resistant NTHi isolates remains low. The nalidixic acid disk test is useful for detecting the first changes in GyrA or in GyrA plus ParC among fluoroquinolone-susceptible strains that are at a potential risk for the development of resistance under selective pressure by fluoroquinolone treatment.

scholarly journals Escherichia Coli O157:H7 Outbreak Associated with Consumption of Ground Beef, June–July 2002

2005 ◽  
Vol 120 (2) ◽  
pp. 174-178 ◽  
Author(s):  
Richard L. Vogt ◽  
Laura Dippold
Keyword(s):  
Escherichia Coli ◽  
Case Control Study ◽  
Ground Beef ◽  
Case Control ◽  
Pulse Field ◽  
Environmental Study ◽  
E Coli ◽  
Pulse Field Gel Electrophoresis ◽  
Environmental Investigation ◽  
Control Study

Objective. A case-control and environmental study tested the hypothesis that purchasing and eating ground beef from a specific source was the cause of a cluster of cases of hemolytic uremic syndrome (HUS) and Escherichia coli ( E. coli) O157:H7 gastroenteritis. Methods. A case-control study comparing risk factors was conducted over the telephone on nine case-patients with 23 selected controls. An environmental investigation was conducted that consisted of reviewing beef handling practices at a specific local supermarket and obtaining ground beef samples from the store and two households with case-patients. Results. The analysis of the case-control study showed that eight case-patients (89%) purchased ground beef at Grocery Chain A compared with four controls who did not develop illness (17%) (matched odds ratio=undefined; 95% confidence interval 2.8, ∞; p=0.006). The environmental investigation showed that Grocery Chain A received meat from Meatpacker A. Laboratory analysis of meat samples from Meatpacker A and Grocery Chain A and stool samples from some patients recovered an identical strain of E. coli O157:H7 according to pulse-field gel electrophoresis. Conclusions. Both the case-control and environmental studies showed that purchasing ground beef at Grocery Chain A, which received ground beef from Meatpacker A, was the major risk factor for illness in eight case-patients; the ninth case-patient was found to be unrelated to the outbreak. Furthermore, meat from Meatpacker A was associated with a nationwide outbreak of E. coli O157:H7 illness that resulted in the second largest recall of beef in U.S. history at the time.

Antimicrobial Resistance Profiles and Clonal Relationship among Non-ESBL Avian Pathogenic Escherichia coli Isolates and ESBL Producing E. coli Isolates from Human Urinary Tract Infections

2020 ◽  
Author(s):  
Bahman ABDI-HACHESOO ◽  
Abdollah DERAKHSHANDEH ◽  
Mohammad MOTAMEDIFAR ◽  
Negar AZIMZADEH
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Geographical Area ◽  
Pulse Field ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Pulse Field Gel Electrophoresis ◽  
Pathogenic Escherichia Coli ◽  
Imipenem Resistance ◽  
Tract Infections

Background: We aimed to investigate antimicrobial resistance and clonal relationships among poultry Escherichia coli isolates from different broiler farms and their relationships with Extended-Spectrum Beta-Lactamase (ESBL) producing urinary pathogenic E. coli (UPEC) isolates from the same geographical area. Methods: Twenty four E. coli isolates from six broiler farms with colibacillosis and 97 ESBL producing human UPEC isolates were investigated for resistance to critically important antimicrobials in human medicine in Shiraz, central Iran in 2015-16. In addition, clonal relationships of these isolates were investigated with Pulse Field Gel Electrophoresis (PFGE). Results: As expected, cephalosporins and imipenem resistance were significantly higher in ESBL producing human E. coli isolates in comparison with non-ESBL avian pathogenic E. coli (APEC) isolates. In addition, significantly higher percentages of gentamycin and trimethoprim-sulfamethoxazole resistance were seen in human isolates. In contrast, nitrofurantoin resistance was significantly higher in APEC isolates. Based on PFGE patterns, five clusters were identified in APEC isolates. Isolates from each farm were closely related to each other by PFGE patterns. However, different PFGE restriction profiles were seen among the E. coli isolates from different broiler farms. Comparison of PFGE patterns among APEC and UPEC isolates showed two closely related PFGE patterns. Conclusion: There were clonally related E. coli isolates caused the outbreaks of colibacillosis within broiler farms. Some of these isolates had closely related PFGE patterns with human UPEC isolates which suggest avian pathogenic E. coli strains as a potential zoonosis.

scholarly journals Manganese Is Required for the Rapid Recovery of DNA Synthesis following Oxidative Challenge in Escherichia coli

2019 ◽  
Vol 201 (24) ◽  
Author(s):  
Corinne R. Hutfilz ◽  
Natalie E. Wang ◽  
Chettar A. Hoff ◽  
Jessica A. Lee ◽  
Brandy J. Hackert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Synthesis ◽  
Genomic Dna ◽  
Rapid Recovery ◽  
Content Type ◽  
E Coli ◽  
Oxidative Challenge ◽  
Alternative Enzymes ◽  
Iron And Manganese

ABSTRACT Divalent metals such as iron and manganese play an important role in the cellular response to oxidative challenges and are required as cofactors by many enzymes. However, how these metals affect replication after oxidative challenge is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. We show that the manganese-dependent recovery of DNA synthesis occurs independent of lesion repair, modestly improves cell survival, and is associated with elevated rates of mutagenesis. The Mn-dependent mutagenesis involves both replicative and translesion polymerases and requires prior disruption by H2O2 to occur. Taking these findings together, we propose that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. The data suggest that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity. IMPORTANCE Iron and manganese play important roles in how cell’s cope with oxygen stress. However, how these metals affect the ability of cells to replicate after oxidative challenges is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. The manganese-dependent recovery of DNA synthesis occurs independently of lesion repair and modestly improves survival, but it also increases the mutation rate in cells. The results imply that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. We propose that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.

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