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Escherichia coli

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Emma Blackwood

on 13 April 2015

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Transcript of Escherichia coli

Category
Domain
Phylum
Class
Order
Family
Genus
Species
Taxonomy
Escherichia coli
Gram Negative, Non-spore Forming, Rod
Microbial Anatomy
As a Facultative Anaerobe
E. Coli
is capable of switching from aerobic to anaerobic respiration, although anaerobic respiration is preferred.
As a chemoheterotroph
E. Coli
uses organic compounds ie. Glucose as a carbon source. More specifically this is a chemoorganoheterotroph.
Microbial Physiological
Highlights
Metabolism
E. Coli
can be grown and cultured very easily in a laboratory setting and thus has been intensively studied for over 60 years. Making it the most widely studied prokaryotic model organism.
The Model Organism
Growth Requirements
Environment
Publication of the Entire E. coli (K12) genome was completed in 1997 by Blattner, Plunkett and colegues.
Genetics
Prokaryotic
Bacteria
Proteobacteria
Gammaproteobacteria
Enterobacteriales
Enterobacteriaceae
Escherichia
coli
Total Strains: 72 Human Strains
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E.Coli 's
claim to fame
Current Research Highlight
van Summeren-Wesenhagen and Marienhagen have developed a platform strain of E.Coli utilizing the ability of E. Coli to readily take up plasmids and it's viability in the lab. This strain will produce pinoslyvin at high concentrations in a more environmentally friendly and cost effective way.
They constructed different bio-synthetic pathways from a set of two different enzymes using isolated genes from various species of
Pinus
plants. These genes were recombined into
E.Coli
BL21(DE3) for production of protein. After optimizing the gene expression in the pathways low concentrations of pinoslyvin were detected.
They then approached the growth factors and precursors necessary for the construction of pinoslyvin,
as well as the pathway intermediates. They were able to isolate the two bottlenecks in the
procedures and from there optimized the pathway with the addition of cerulenin (an antibiotic)
leading to the production of 70mg/L of pinoslyvin from glucose.
Finally they further increased the titers with the addition of L-phenylalanine to 91mg/L.
Which far out measures the 0.6mg/L achieved from supplemented
Streptomyces venezuelae
.
Summary
References
Other Fun Facts
E. Coli was named after Theodor Escherich who isolated the types of species in this genus
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Background Image: Rocky Mountain Laboratories, NIAID, NIH - NIAID: These high-resolution (300 dpi) images may be downloaded directly from this site. All the images, except specified ones from the World Health Organization (WHO), are in the public domain. For the public domain images, there is no copyright, no permission required, and no charge for their use.
Found on Wikipedia
1.
Ahmer, M.M.B.
2004. Cell-to-cell signaling in Escherichia coli and Salmonella enterica. Molecular Microbiology. 52: p. 933-945.
2.
Berg, C. H.
2010. Bacterial motility and behavior. Rowland Institute for Science. Cambridge, MA. http://www.rowland.harvard.edu/labs/bacteria/movies/swarmecoli.php.
3.
Berg, C. H
. 2004. E. Coli in motion, p. 2,3,49,69, 105. Of the Biological and medical physics biomedical engineering series. Springer-Verlag New York Inc, New York, NY.
4.
Blattner, F. R., Plunkett G. III, Bloch C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C. K., Mayhew, F. G., Gregor, J., Davis N.W., Kirkpatrick, H. A., Goeden, M.A., Rose, J.D., Mau, B., Shao, Y.
1997. The complete genome sequence of Escherichia coli K-12. Science. 277: p 1453 -1462.
5.
Brown, A. D, Berg, C. H.
1974. Temporal stimulation of chemotaxis in Escherichia coli. Proc Nat Acad. Sci. 71:1388-1392.
6.
Chapman, M. R., Robinson, L. S., Pinkner, J. S., Roth, R., Heuser, J., Hammar, M., Normark, S., Hultgren, S. J.
2002. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science. 295: p. 851-855.
7.
Expression Technologie
s http://www.exptec.com/Expression%20Technologies/Bacteria%20growth%20media.htm#Nutrient_contents_in_commonly_used_bacterial_E.coli_growth_media
8.
Goldman E.. Green L.H..
2009. Practical Handbook of Microbiology 2nd edition. P. 73,74,218,219. Taylor & Francis Group, Boca Raton, FL.
9.
Hong Kim, B., Gadd, G.M.
2008. Bacterial physiology and metabolism, p. 61- 92. Cambridge University Press, Melbourne, NY.
10.
Kayser, F.H., Bienz, K.A., Eckert, J., Zinkernagel, R.M.
2005. Medical microbiology, p. 223. Thieme, New York, NY.
11.
Microbial Genetics
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/rev-sup/wobble.html
12.
Microbe Wiki
https://microbewiki.kenyon.edu/index.php/Escherichia_coli
13.
Rudolph, R., Lilie, H.
1996. In vitro folding of inclusion body proteins. The FASEB Journal. 10: p 49-56.
14.
Sankaran, N.
Microbes and People: The A – Z of microorganisms in our lives, p. 15, 100- 101, 139-140. Oryx Press Pheonix, AZ.
15.
Scheutz, F., Strockbine, N.A
.. 2005. The proteobacteria, part b: the gammaproteobacteria. Bergey’s Manual of Systematic Bacteriology. p: 607 - ______.
16.
Than, K.
2011. Bacteria grow under 400,000 times earth’s gravity. National Geographic News. http://news.nationalgeographic.com/news/2011/04/110425-gravity-extreme-bacteria-e-coli-alien-life-space-science/
17.
van Summeren-Wesenhagen, P.V., Marienhagen, J.
. 2015. Metabolic engineering of Escherichia coli for the synthesis of plant polyphenol pinoslyvin. Applied and environmental microbiology. 81: p. 840-849. Received From: American Society for Microbiology, http://journals.asm.org/ .
18.
Versalovic J.. Carroll K.C.. Jorgensen J.H.. Funke G.. Landry M.L.. Warnock D.W.
. Manual of Clinical Microbiology 10th edition. Volume 1. P. 643, 644, 651, 1095. American Society for Microbiology, Washington, DC.

There are a total of 5 species under the genus Escherichia; E. coli, E. hermannii, E. Fergusonii, E. vulneris, and E. blattae
Motility
There are as many as 10 E.Coli cells in 1 cubic cm of human stool.
10
3
Three Layers to the Cell Wall
Thin Outer Membrane
Lipopolysaccharides with outward facing sugar chains, penetrated by porins
Blocks lipid soluble, allows water soluble passage.
Peptidoglycan Porous layer
provides rigity and cell structure
Immersed in periplasm
Cytoplasmic - Phospholipid - layer
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Flagellar Motors are powered by protonmotive force created by protons moving down an electrochemical gradient.
3
11 are involved in Human Disease, Enterovirulent E. coli (EEC), further differentiated to 6 classes
16 other Strains found in other Mammals
The Strains are differentiated in Groups based on the O (Lipopolysaccharide), K (capsule), H (flagellum) antigens possessed by the strain.
The Most Well Known Microbe
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Average Genome Size: 4.6 million base pairs which create around 4,300 genes. Arranged in a cyclic strand free in the cytoplasm
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Plasmids
Some strains contain F Factor in the plasmid or chromosome, allowing transfer of genes through conjugation.
This also allows for horizontal gene transfer through generations.
E. Coli is able to take up antibiotic resistant genes, such as ampicillin.
Or genes that encode for the production of green fluorescent
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Many strains of are motile
E.Coli
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Motile Strains have peritrichous flagella
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Fimbriae allow adhesion to the intestinal wall.
Specific strains also possess Pili which allow for conjugation
E. Coli
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https://www.bio.cmu.edu/courses/03441/TermPapers/2001TermPapers/chemotaxis/caroline.html
Runs - movement away or towards a stimulus
Counter Clockwise Flagellar Rotation
Causing the multiple flagellum to move in a synchronized fashion
Tumbles - random "Tumbling" change of direction
Clockwise un-synchronized Flagellar Rotation

(Projecting in all directions)
exhibit chemotaxis based on the temporal sensing mechanism (sensing the stimulus change over a short period of time ~ 10s )
Therefore is able to direct it's motion towards (positive chemotaxis) stimuli such as nutrients or amino acids.
E.Coli
E.Coli
3
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Direction of rotation is controlled by intracellular signals, that are generated by receptors embedded in the cell wall. These receptors count molecules of interest, the more molecules the stronger the signal, with a strong enough signal the flagellum will rotate and the cell will move.
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Movement In a Group or "Swarm"
Cells are seen to "swirl" in packs or rafts, uniformly, the edges of a swarm form a single layer of cells that are relatively immobile - almost pushed by other cells. While in the centre cells form multple layers of very actively mobile cells.
See 1s-10s of Video.
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2
2
E. Coli
is typically found singularly or in pairs
Other Structures:
http://watcut.uwaterloo.ca/webnotes/Pharmacology/microbesBacterialCellWall.html
Inclusion Bodies

Found in both the cytoplasmic and periplasmic space
Used in inductrial processes where E. Coli creates a protein which forms inclusion bodies and is then extracted
Even complex proteins can be successfully folded and extracted within
E. Coli
Highly hydrophobic protein is more prone to accumulate as inclusion bodies
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E. Coli is the most frequent human pathogen.
4
Curli
Amyloid fibres that produced by E. Coli through unique nucleation precipitation cellular machinery and found in their complex cellular matrix.
Help mediate;
cell adhesion, invasion, aggregation and biofilm formation.
Potent inducers of inflammatory responses in hosts.
Responsible for human diseases such as Alzheimer's, and Huntington's
E. Coli provide an excellent medium for the study of these fibers
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http://sites.lsa.umich.edu/chapman-lab/
Aerobic Pathways Examples
Anaerobic Pathways Examples
Embden–Meyerhof–Parnas
Enter-Doudoroff
Modified EMP Pathway
Phosphate Limited Conditions
E. Coli will oxidize dihyroxyacetone phosphate through methylglyoxal to pyruvate.
Allows continuation of Acetyl-CoA sythesis when G3P dehydrogenase is inactive
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Hexose monophospate pathway (HMP)
When glucose is the sole carbon source
72% is used through EMP and 28% through HMP to fulfill the requirements for biosynthesis.
Some eukaryotes use HMP to metabolize glucose.
Step 1) glucose-6-phosphate is oxidized to ribulose-5-phospate coupled to reduction of NADPH
Step 2) Ribulose-5-phosphate is converted to ribose-5-phosphate and xylulose-5-phosphate.
Step 3) pentose-5-phosphates are rearranged to glucose-6-phosphate and glyceraldehyde-3-phosphate (G3P).
Incomplete Tricarboxylic Acid Cycle Fork
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Serves as a host organism for the majority of work with recombinant DNA. E. Coli plays an important role in the rapid continuous production of necessary proteins.
(Even second year university students can culture it!)
Model Organism def:
"a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the working of other organism"
http://www.telegraph.co.uk/news/health/news/8543309/Cucumber-E.Coli-outbreak-kills-five-in-Germany-and-leaves-three-ill-in-UK.html
E. coli
has been manipulated to produce human insulin.
See the Research Highlight for another example of this.
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Optimal Growth*
Mixed-Acid Fermentation
Temp: mesophilic, 15 - 45 °C, 37 °C*

pH: neutrophilic, 5.0 to 9.0
Salt Tolerant
Carbon Source: Glucose*, Lactose
some strains as low as 7.5°C and as high as 49°C
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Commonly Used Medias
Naturally part of the common microflora of the large intestine of humans it is also found in the gut of other mammals.
E. Coli
is often found with hydrogen-consuming organisms since the fermentation pathways of E. coli produce hydrogen gas.
Enteropathogenic
E. coli
can also be found in stagnant water that has been contaminated with feces, and undercooked meat. Which is why it poses such a risk to Humans who ingest these
http://www.alargastro.com/Patient_Education/Large_Intestine.html
Some Unique Metabolic Features
Recently Shigeru Deguchi and colleagues successfully cultivated
E. Coli
at hypergravity (400,000 x Earth's Gravity).
The E. Coli lumped together but multiplied normally
Attributed to the size, and lack of organized organelles.
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Extreme E.Coli
Identification
Summary
Background
Results
We are always looking for a way to produce things in a less expensive and more environmentally safe way. But how do we approach that when we are producing something we need? Plant pholyphenols specifically pinoslyvin is effective in the treatment of cardiovascular disease, cancers, as well as arthritis. But currently it is really expensive to manufacture. That is where E. Coli comes in. The researchers in this project set out to turn a strain of
E. Coli
into a pinoslyvin factory, to lower the production cost and ultimately the price of the medication it is used in.
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all information unless otherwise stated is from Ref 12.
http://www.fz-juelich.de/SharedDocs/Personen/IBG/IBG-1/EN/Research_groups/senseup/van_summeren_wesenhagen.html?nn=547728
http://www.fz-juelich.de/SharedDocs/Personen/IBG/IBG-1/EN/Research_groups/syncell/marienhagen.html
http://www.snipview.com/q/Pinosylvin
They found that (shown below) that with the supplementation of L-phenylalanine and the addition cerulenin the E.Coli produced the highest yield of pinoslyvin.
Then they determined the optimal modifications to E.Coli genome that will (with the addition of L- phenylalanine and cerulenin) yeild the highest pinoslyvin.
These results demonstrated that 200microMcerulenin and 3miliM L-phe in the prescence of E.Coli BL21(DE3)/pR-HisPstrsts2 with the T248A substitution was the optimal choice.
1
2
van Summeren-Wesenhagen and Marienhagen were able to manipulate
E.Coli
to produce Pinoslyvin with high yields.
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all information unless otherwise stated is from Ref 12.
Quorum Sensing
E.Coli being a gram neg. bacteria uses Quorum sensing as cell to cell communication to regulate things like cell division.
1
E.Coli is extremely viable in the lab, with only a 20 minute generation time, providing quick and easy culturing.
It typically colonizes an infant's GI tract within hours of birth.
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The detection of B-Glucuronidase activity is useful for the rapid identification of E. Coli.
Through a colorimetric (ex. p-nitrophenyl-B-D-glucopyranoside) or fluorometric substrate (ex. MUG)
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Detection of B-Galactosidase activity is an identification of Enterobacteriaceae
o-nitrophenyl-B-D-galatopyranoside (ONPG) being used in the place of lactose causes bacteria to release o-nitrophenol
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MacConkey Agar
LB Broth
SOB/SOC medium
TB/SB medium
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Will oxidize pyruvic acid, formic acid, hydrogen, and amino acids. Paired with the reduction of oxygen, nitrate, fumarate, dimethyl suloxide and trimethylamine N- oxide
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The fermentation of lactose of E.Coli will develop into purple colonies on MacConkey Agar as neutral red indicator is affected by the acids produced.
E.Coli will completely clear the red in blood agar tests.
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Naturally occurring E.Coli does not require any additional growth factors, as it is able to manufacture what it needs.
Although adding the following will encourage growth;
Peptone or Tryptone, Yeast Extract, Sodium Chloride, Magnesium, Potassium, Phosphate, Glycerol
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It is very easy for E.Coli to take up extraneous DNA and RNA fragments. This is known as Natural Transformation.
Thus creating great diversity amongst E.Coli and contributing to its usefulness in research and manufacturing
protein, example on the pGreen plasmid.
http://www.web-books.com/MoBio/Free/Ch3H3.htm
There are 86 tRNA genes on the E. coli chromosome.
only 47 different tRNAs are required to recognize all the possible codons
Escherichia coli from the family Enterobacteriaceae
Gram Negative Rod Bacteria
With Fimbrae and sometimes Pili, inclusion bodies.
Motile with the use of peritrichious flagellum
Utilizes Cruli and Quorum sensing.
Detected by purple colonies on MacConkey Agar, and clearing of the red on blood agar plates.
Faculatative Anaerobe
Mesophile, Neutrophile
Normally found in the human GI tract
Model Organism
Very viable in a laboratory
Easily takes in plasmids for recombination techniques.
Natural Transformation creates many strains
Used in manufacturing of many proteins
Ex. insulin, pinoslyvin.
http://www.cdc.gov/pulsenet/pathogens/ecoli.html
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In 1999 it was estimated that about 73,000 people in the U.S. got sick each year from E. coli
But only 60 died from these infections.
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Please consider watching the prezi online, to see animations and video. http://prezi.com/x8kpkvlzr5jq/?utm_campaign=share&utm_medium=copy
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