Julia Anderson
Classen School of Advanced Studies
Abstract
Gram negative bacteria require iron for cellular respiration. It has been proposed that
iron uptake prevention antibiotics can be developed to inhibit the iron transport protein
Fep A. Ferric enterobactin was purified and crystallized for use in research concerning
FepA's function in the outer membrane of gram-negative bacteria. The gram-negative
bacteria Escherichia coli was grown in T-Media. Ethyl acetate extractions were performed,
and the ethyl acetate extract was concentrated and crystallized. The average yield of
enterobactin per trial was 1.1647 grams. The purification procedure is time-dependent, and
higher yields of enterobactin result from more expedient work. The enterobactin produced
during this project will be used for research concerning the development of iron uptake
prevention antibiotics.
Introduction
Gram-negative bacteria are responsible for shigellosis, typhoid fever, salmonellosis,
traveler's diarrhea, and cholera1. While Gram-negative bacteria account for only a small
portion of the total number of bacterial species, gram-negative bacteria cause a
disproportionate number of diseases2. In spite of the human body's natural defense
efforts, bacterial infections are often harmful and sometimes fatal. Therefore, finding
other methods of controlling bacterial infections has been critically important to
maintaining modern standards of health.
Prevention is the most effective strategy of bacterial control. Pure water supplies,
pasteurization of milk, and sterilizing medical utensils has greatly reduced the
transmission of bacterial diseases in industrialized countries. Enhancing immunity against
bacterial infection by vaccination is another standard method of controlling disease. A
third method of bacterial disease prevention is the application of chemotherapy.
The treatment of bacterial infections with antimicrobial agents became common in the 1940s
during World War II when the discovery of penicillin in the 1940s revolutionized the
treatment of bacterial disease, allowing even critically ill patients to recover. These
antibiotics proved to be the wonder drugs of the mid 20th century; however, bacterial
populations have quickly evolved to resist many established antibiotics. Bacterial
diseases that were once thought conquered with the use of antibiotics are now experiencing
a resurgence in activity.
Purpose
With the emergence of antibiotic resistant strains of gram-negative diseases, it has
become imperative to find new ways of combating bacterial disease. Dr. Philip Klebba's lab
at the University of Oklahoma, along with several other labs worldwide, has been focusing
its research on a protein, Ferric Enterobactin Permease A (Fep A) that is the initial
component of the primary iron transport system in many3. Iron transport through this
system has shown to be essential for cellular respiration.4 Thus, drugs that prevent the
uptake of iron through Fep A would create an entirely new class of antibiotics. One
drawback of this hypothesis is that iron transport is also essential for human tissues, so
the new antibiotics will have to be highly specific for the bacterial system.5
The purpose of my research was to purify ferric enterobactin to investigate outer membrane
protein structure and function in Escherichia coli. Gram-negative bacteria such as E. coli
obtain iron for cell energy by using a siderophore called ferric enterobactin.
Enterobactin exits the cell and chelates the first iron molecule it encounters. The
enterobactin returns the iron to the cell through a protein receptor located in the cell
membrane called Ferric Enterobactin Permease A. To study how FepA interacts with
enterobactin, the enterobactin must be purified and crystallized before experimentation
can proceed.
Pertinent Scientific Literature
Researchers at the University of Oklahoma, working in the lab of Dr. Phillip E. Klebba
have published many articles concerning their research in outer membrane protein structure
and function. In the May 23, 1997 issue of Science, Dr. Klebba and his group reported that
the membranes of many gram-negative bacteria admit iron (iron is necessary for the
organisms to become pathogenic) through protein-gated pores that open and close6. These
pores are found in such highly dangerous gram-negative bacteria as those that cause
cholera, dysentery, blood poisoning, meningitis, and plague.
The Science paper shows that bacteria bind iron-containing molecules on the outside of a
closed membrane protein, called FepA. After binding iron, the protein opens and then
closes as it transports the metal into the cell. Now that the mechanisms by which the
pores operate are known, researchers can proceed to study new approaches for fighting the
pathogens, including ways to manipulate the gated pores to allow drugs to enter or to
prevent iron from entering the cells.
In 1998, Susan Buchanan and Barbara Smith, from the University of Texas Southwestern
Medical Center and Dick van der Helm from the University of Oklahoma discovered the
crystal structure of the outer membrane protein FepA from E. coli and isolated and
purified FepA in the presence of a detergent. Crystals were grown from solutions
containing detergent and polyethylene glycol7.
In a second paper recently published in the Proceedings of the National Academy of
Sciences, the University of Oklahoma researchers found that iron-containing molecules
attach to the surfaces of cells by binding to the outside of a closed membrane protein.
After binding iron, the protein opens to internalize the metal.
In addition, the experiments identify a methodology -- electron spin resonance
spectroscopy -- suitable for studying these events in living cells. Living cells can be
labeled and studied with this approach without disrupting their natural. The research thus
makes it possible to observe transport events as they happen, which will lead to new
insights about the molecular mechanisms of membrane transport.
Methodology
The procedure for the purification of enterobactin was carried out under the supervision
of Dr. Phillip Klebba and his graduate students at the University of Oklahoma. Laboratory
protocols and safety techniques were enforced, as it is unsafe for inexperienced students
to work unsupervised in a working biochemical lab. As the techniques became more familiar
to us the supervision of the enterobactin purification process became more relaxed.
Eventually, the procedure was more or less carried out on our own, however, Dr. Klebba and
his research team were always close at hand to answer any questions or assist in the
operation of unfamiliar lab equipment.
T-Media is used in the laboratory as a medium for growing E. coli. Prepare 15 L T-Media.
The E.coli must be grown separately before being subcultured to the T-Media. To do this,
inoculate the strain AN102 in 5 mL LB broth and add 50 ?L streptomycin. After bacteria has
grown 12-14 hours subculture to 150 mL LB broth and add 1.5-mL streptomycin and incubate
for 8 to 10 hours. Inoculate bacteria in 15 L T-Media and grow over night at approximately
34 0C overnight in water bath for 15 to 16 hours.
After 16 hours, weigh out equal amounts of T-Media solution into 1-L centrifuge bottles
and spin down for 40 minutes at 4000 rpm in the J6-HC centrifuge. Save the supernatant in
flask and dispose of sedimentary liquid. Repeat for all 15 L of the T-Media solution.
Place 1-L of the supernatant in a separatory funnel. Perform two ethyl acetate
extractions: 1x 200 mL; 1x 100mL. When extracting wait a couple minutes for the two
layers to separate. Drain off the bottom layer and place top layer in another flask.
Repeat extractions for all 15 L of supernatant. Concentrate the extract by roto vaporizing
down to a 100-mL volume. Transfer ethyl acetate extract to a new container and extract
once with 100mM citrate, pH 5.5. Extract once with 10-mL dH2O. Add extract to 1-L flask
and dry 8-12 hours in anhydrous MgSO4.
Filter away MgSO4 into a dry container. Save ethyl acetate extract in flask. Dispose of
MgSO4. Roto vapor total volume to 50 ml in 1L teardrop flask and then concentrate to
approximately 10 ml in 100 ml tear drop flask. Add hexanes slowly until crystals form and
then centrifuge at 4,000 rpms for 5 minutes. Place product in desiccator until completely
all liquid has been removed.
Outcomes
The procedure for purifying and crystallizing ferric enterobactin was repeated six times.
Yields were 1.4355 g; 1.1387 g; 0.9462 g; 1.3682 g; 0.8275 g; and 1.2721-g enterobactin
for trials one through six, respectively. The enterobactin is essential to the research in
the lab of Dr. Klebba. The research in the lab focuses on the ferric enterobactin receptor
protein FepA and its role in the outer membrane function of gram-negative bacteria.
Conclusions
The purification of ferric enterobactin is a procedure that is vital for the study of
outer membrane function in gram-negative bacteria. The function of the ferric enterobactin
receptor FepA can only be studied by observing the transport of ferric enterobactin across
the membrane, so it is necessary to have purified enterobactin ready for use. The
procedure for purifying ferric enterobactin is a time-dependent procedure. We found that
more enterobactin was produced when the procedure was carried out in an expedient manner.
It is more efficient to have a higher yield of purified enterobactin per trial, however,
time constraints, equipment malfunction, and human error create obstacles that have a
direct effect on the amount of enterobactin produced per trial. While the yields of
enterobactin produced varied somewhat the consistency of the amount of enterobactin
produced is not as important as the purity.
Iron uptake prevention antibiotics may prove to be an effective agent against bacterial
scourges, but their realization will be some years off. While research using the ferric
enterobactin produced in this experiment and the function of Fep A and Fep A's role in
iron transport in the cell continues, current antibiotics are being used to control
diseases despite setbacks such as toxicity to human cells and, most devastatingly,
antimicrobial resistance. The rate at which bacterial cells evolve allows bacteria to
acquire resistance to certain antibiotics in a matter of a few months or years. Because
bacteria acquire antibiotic resistance so quickly, it has become increasingly important to
develop these iron uptake prevention antibiotics.
Appendix
PREPARATION OF T-MEDIA
NaCl 87 g
KCl 55.5 g
NH4Cl 16.5 g
CaCl2 2H2O 2.25 g
MgCl2 6H2O 1.5 g
Na2SO4 2.13 g
KH2PO4 4.08 g
Tris base 181.5 g
Dissolve in 3 liters distilled water and add 115 mL HCl. Adjust pH to 7.4. Dilute to 15 L.
Autoclave T-Media.
After autoclaving, add:
Leucine 0.6 g
Proline 0.6 g
Tryptophan 0.75 g
B1 0.0375 g
Casamino acids (10%) 10 mL
MnSO4 H2O (1mM) 1.5 mL
Glucose (20%) 300 mL
Streptomycin 1.5 g
Ampicillin 1.5 g
1 Black, Jacquelyn G. Microbiology. Englewood:Simon and Schuster. 1993.
2. Microbionet. Gram negative bacteria. [Online] Available
http://www.sciencenet.com.au/frames/profiles/negative/f-negat.htm October 8, 1999
3 Bettelheim, Karl, and Gavin Thomas. E. coli irom acquisition. [Online] Available
http://sun1.bham.ac.uk/bcm4ght6/path/irona.html. August 12, 1999
4 Klug, Candice and Jimmy B. Feix. Towards development of bacterial iron transport antibiotics. [Online] Available http://www.biophysics.mcw.edu/bri-epr/HIGHLIT.html. July 23, 1999
5 Klug, Candice and Jimmy B. Feix. Towards development of bacterial iron transport antibiotics. [Online] Available http://www.biophysics.mcw.edu/bri-epr/HIGHLIT.html. July 23, 1999
6 Jiang, X., M.A. Payne, Z. Cao, S.B. Forester, J.B. Feix, S.M.C. Newton & P.E.
Klebba. 1997. Ligand-specific opening of a gated-porin channel in the outer membrane of
living bacteria. Science 276: 1261-1264
7 Smith, B.S., Kobe, B., Kurumbail, R., Buchanan, S.K., Venkatramani, L., van der Helm,
D. & Deisenhofer, J. 1998. Crystallization and preliminary x-ray analysis of ferric
enterobactin receptor FepA, an integral membrane protein from Escherichia coli. Acta.
Cryst. D., in press.
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Julia Anderson : Purification of Ferric Enterobactin
