scholarly journals Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae

2019 ◽  
Vol 20 (20) ◽  
pp. 5212 ◽  
Author(s):  
Maximillian P. M. Soltysiak ◽  
Rebecca S. Meaney ◽  
Samir Hamadache ◽  
Preetam Janakirama ◽  
David R. Edgell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Donor Cell ◽  
Dna Delivery ◽  
Dna Transfer ◽  
Solid Media ◽  
Wide Range ◽  
Semi Solid ◽  
Plasmid Size ◽  
Synthesis Protocol

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

scholarly journals Trans-Kingdom Conjugation Within Solid Media from Escherichia coli to Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Maximillian P.M. Soltysiak ◽  
Rebecca S. Meaney ◽  
Samir Hamadache ◽  
Preetam Janakirama ◽  
David R. Edgell ◽  
...  
Keyword(s):  
Donor Cell ◽  
Dna Delivery ◽  
Dna Transfer ◽  
Liquid Media ◽  
E Coli ◽  
Solid Media ◽  
Wide Range ◽  
Semi Solid ◽  
Plasmid Size ◽  
Synthesis Protocol

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from E. coli to S. cerevisiae. We demonstrated that there was no substantial decrease in conjugation frequency as plasmid size increased—up to 138.5 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids

scholarly journals Microbial diversity beyond E. coli: new microbial worlds, new concepts in biology

2011 ◽  
Vol 32 (2) ◽  
pp. 73
Author(s):  
John A Fuerst
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Influenza Virus ◽  
Microbial Diversity ◽  
Microbial Evolution ◽  
E Coli ◽  
New Concepts ◽  
Wide Range ◽  
Classical Models ◽  
The Universe

Microbial diversity explores the universe of microorganisms beyond classical models such as Escherichia coli, influenza virus, or Saccharomyces cerevisiae. Exploring such new microbial worlds is essential for a microbiology which needs to learn about all the scientific and practical possibilities offered by billions of years of microbial evolution. Here we illustrate some examples of how studying a wide range of microbial diversity can assist microbiology as a fundamental and a practical science.

scholarly journals Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

2015 ◽  
Vol 81 (13) ◽  
pp. 4458-4476 ◽  
Author(s):  
Christian Bille Jendresen ◽  
Steen Gustav Stahlhut ◽  
Mingji Li ◽  
Paula Gaspar ◽  
Solvej Siedler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Cinnamic Acid ◽  
Aromatic Compounds ◽  
Physcomitrella Patens ◽  
Coumaric Acid ◽  
Content Type ◽  
Highly Active ◽  
Enhanced Production ◽  
Wide Range

ABSTRACTPhenylalanine and tyrosine ammonia-lyases form cinnamic acid andp-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequencesin silicousing synteny information and aiming for sequence divergence. We performed a comparativein vivostudy, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production ofp-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacteriumBrevibacillus laterosporus, a bacterial-type enzyme from the amoebaDictyostelium discoideum, and a phenylalanine ammonia-lyase from the mossPhyscomitrella patens(producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium usingEscherichia colias the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes fromHerpetosiphon aurantiacusandFlavobacterium johnsoniaeresulted in high production ofp-coumaric acid inEscherichia coli(producing 440 μMp-coumaric acid OD600unit−1in the medium) and inLactococcus lactis. The enzymes were also efficient inSaccharomyces cerevisiae, wherep-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

scholarly journals Saccharomyces cerevisiae displays an increased growth rate and an extended replicative lifespan when grown under respiratory conditions in the presence of bacteria

2017 ◽  
Vol 63 (9) ◽  
pp. 806-810
Author(s):  
Paul A. Kirchman ◽  
Nicholas Van Zee
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Volatile Compound ◽  
Lifespan Extension ◽  
Yeast Saccharomyces Cerevisiae ◽  
Replicative Lifespan ◽  
Solid Media ◽  
Respiratory Conditions ◽  
Ferment Glucose

Individual cells of the budding yeast Saccharomyces cerevisiae have a limited replicative potential, referred to as the replicative lifespan. We have found that both the growth rate and average replicative lifespan of S. cerevisiae cells are greatly increased in the presence of a variety of bacteria. The growth and lifespan effects are not observable when yeast are allowed to ferment glucose but are only notable on solid media when yeast are forced to respire due to the lack of a fermentable carbon source. Growth near strains of Escherichia coli containing deletions of genes needed for the production of compounds used for quorum sensing or for the production of the siderophore enterobactin also still induced the lifespan extension in yeast. Furthermore, the bacterially induced increases in growth rate and lifespan occur even across gaps in the growth medium, indicating that the bacteria are influencing the yeast through the action of a volatile compound.

scholarly journals Short-Term Pretreatment of Sub-Inhibitory Concentrations of Gentamycin Inhibits the Swarming Motility of Escherichia Coli by Down-Regulating the Succinate Dehydrogenase Gene

2016 ◽  
Vol 39 (4) ◽  
pp. 1307-1316 ◽  
Author(s):  
Yijing Zhuang ◽  
Weidong Chen ◽  
Fen Yao ◽  
Yuanchun Huang ◽  
Shuqin Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Succinate Dehydrogenase ◽  
Infectious Agent ◽  
Short Term ◽  
Swarming Motility ◽  
Swimming Motility ◽  
E Coli ◽  
Solid Media ◽  
Antibiotic Effect ◽  
Semi Solid

Background/Aims: Motility is a feature of many pathogens that contributes to the migration and dispersion of the infectious agent. Whether gentamycin has a post-antibiotic effect (PAE) on the swarming and swimming motility of Escherichia coli (E. coli) remains unknown. In this study, we aimed to examine whether short-term pretreatment of sub-inhibitory concentrations of gentamycin alter motility of E. coli and the mechanisms involved therein. Methods: After exposure to sub-inhibitory concentrations (0.8 μg/ml) of gentamicin, the swarming and swimming motility of E. coli was tested in semi-solid media. Real-time PCR was used to detect the gene expression of succinate dehydrogenase (SDH). The production of SDH and fumarate by E. coli pretreated with or without gentamycin was measured. Fumarate was added to swarming agar to determine whether fumarate could restore the swarming motility of E. coli. Results: After pretreatment of E. coli with sub-inhibitory concentrations of gentamycin, swarming motility was repressed in the absence of growth inhibition. The expression of all four subunits of SDH was down-regulated, and the intracellular concentration of SDH and fumarate, produced by E. coli, were both decreased. Supplementary fumarate could restore the swarming motility inhibited by gentamycin. A selective inhibitor of SDH (propanedioic acid) could strongly repress the swarming motility. Conclusion: Sub-inhibitory concentrations of gentamycin inhibits the swarming motility of E. coli. This effect is mediated by a reduction in cellular fumarate caused by down-regulation of SDH. Gentamycin may be advantageous for treatment of E. coli infections.

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

Sign in / Sign up

Export Citation Format

Share Document