1. Escherichia coli: An Analysis of T1 phage on Escherichia coli and classification of
Escherichia coli mutations
University of Mount Union Biology Department
Presley Mays

2. Abstract
The purpose of this experiment was a two part objective. The first was to generate
Escherichia coli that are resistant to T1 bacteriophage and the second was to classifythe
Escherichia coli mutations that result followingresistanceto infection usingPCR. This was
carriedout through two trials of plaque assay with new mutants, regrowthof mutants from
Shaigan Bhatti’s previous research, MRVP confirmationof mutants generatedinprevious
research, and isolation/analysis of DNAthrough PCR. The plaque assay produced
inconclusive results as a significant number of plaques was not generatedin either trial.
However, the regrowthof mutants and MRVP testingof prior researchwere bothsuccessful,
proving that these mutants were in fact Escherichia coli and not a contamination. The last
portionof the experiment is still inprogress but genomic DNA from positive cultures was
isolatedand will be PCR amplified. The DNA will be separatedand analyzed using agarose
gel electrophoresis.
Introduction
Escherichia coli is afrequent cause of life-threateningbloodstream infections and
other common illnesses, suchas urinary tract infections. (Colignon2009). It accounts for
17.3% of clinical infections requiringhospitalizationandis the second most commonsource
of illness (BroadInstitute 2010). Among outpatient infections, Escherichiacoli is the most
commondisease-causing organism (38.6%) Escherichia coli has several distinct types of
bacteriophage that infect it: T1, T2, T3 and T5 coliphage. More specifically, T1
bacteriophage is an enterovirus that causes lysis of Escherichia coli. It multiplies rapidlyand

3. is the only one of the phages that requires an energizedcell membrane for irreversible
binding. (Streips & Yasbin 2002) Althoughthere is various published work onT1, it is one
of the least understoodphages. More knowledge is necessaryas mutations in Escherichia
coli allow it to be resistant to bacteriophage, like T1, which is a major problem in healthcare
(Anderson 2013).
Illness and death associatedwith infectious diseasesinanimals and humans has
greatlydecreaseddue to the crucial role of antimicrobial drugs. However, antibiotics that
were once effective incuring infections do not always work anymore. Antibiotic resistance
can be definedas bacteria’s ability to endure and survive the antimicrobial effects of
antibiotics (Anderson2013). It is a growing global issue as major increases inthe emergence
and spread of drug-resistant bacteriahave occurredover the last two decades. The US Center
for Disease Control andPrevention(CDC) considers antibioticresistance one of their
uppermost concerns as infections of drug-resistant bacteria caneasilylead to larger hospital
expenses and increasedriskof death. The reportedriseof antibiotic resistance is amain
concernfor animals and humans and has beena persistent problem withdrugs suchas
extended-spectrum β-lactams andfluoroquinolones (FQs). This can be explained by drug-
resistant commensal Escherichia coli isolates that institute asubstantial reservoir of
antibiotic resistance determinants. These determinants thenspreadto bacteriapathogenic for
humans and animals. However, this is just one instance. Escherichia coli mutants are
continuallyincreasingantibiotic resistance across the globe.
Data shows that the highest resistancein Escherichiacoli is amongthe antimicrobial
drugs that have been usedfor the longest amount of time. “Surveillance studies conductedin

4. different countries generallyreport anincrease inthe level of resistance in Escherichia coli
isolates to major classes of antibiotics usedfor the treatment of livestockand companion
animals. It has been suggestedthat an important factor inthe emergence and dissemination
of resistance is the selective pressure exertedfollowingantibiotic exposure.”(Okeke 2000)
This becomes aconcernas preventable and treatable illnessescansuddenly become
incurable.
Consequently, the primary goal of this experiment had two parts. The first was to
generate Escherichiacoli that are resistant to T1 bacteriophage and the second was to
classify Escherichia coli mutations, boththose that were grown in this experiment and those
that were grown from previous research. Since this is a fairly new fieldof research, this will
accomplisha better understandingof antibiotic resistance whichcan lead to advances in
medicine and preventionof mutants. Escherichia coli was chosenas it is one of the most
commondiseases and consequentlyone of the topconcerns for antibioticresistance (Broad
Institute 2010).
Background
Mutants of Escherichia coli were isolated ina study by Karczmarczyk in 2011. The
receptor forthe T5 phage on Escherichia coli is the FhuA gene product. This study examined
the biochemical structure of this FhuA gene product to compare and contrast mutations in
Escherichia coli. The results showedthat the deletionof the TonB box regiononthe FhuA
gene structure completelyinactivatedall TonB-dependent functions of FhuA, seenin Figure
1. Fixationof the corkto turn the barrel through a disulfide bridge betweenintroduced
residues eliminatedferrichrometransport. However it was restoredbyreductionof the

5. disulfide bond. It is concludedthat the TonB box is essential for FhuA activity. The TonB
box regionhas to be flexible, but its distance from the corkdomain can greatly vary. The
removal of salt bridges between the corkand the barrel affects the structure but not the
functionof FhuA (Karczmarczyk 2011).
Figure 1. The structure and the mutations introducedinthe FhuA gene at the N-terminus.
The arrows indicate the start and stopsites for the TonB box and switchhelix. The numbers
show the positions of the amino acid residues (Karczmarczyk2011).
Another study in the 1950s by Weidel and Kellenberger experimentedwithlysates of
T5 bacteriophage-infectedcells. This experiment attemptedto isolate T5 receptorsfrom T5-
infectedcells. Receptors were isolatedfrom Escherichia coli. Theyobserved that these cells
containedstructures that were morphologicallyidentical to T5 receptors. However, these
receptorsdidnot bind any of the T5 in the lysate. In Figure 2 it is seenthat receptors isolated
from uninfectedcells causedinactivationof the phage and were observedto attach to the tips
of T5 tails. Weidel and Kellenberger concludedfrom this experiment that the phage could

6. have a mechanism to inactivate the receptorseitherbeforeor at the instant lysis takes place.
It is typicallyassumed that lysates of T5 must be purifiedby centrifugation. This prevents the
phage from being inactivated by the free receptors inthe lysate.
Figure 2. The inactivation of T5 bacteriophage by isolationof receptorsfrom infectedand
uninfectedcells.
(O) Receptors isolatedfrom uninfectedcells;(.) receptorsisolatedfrom cells infectedfor 15
minutes with T5H23c;(□) receptors isolatedfrom cellsinfectedfor 30 minuteswith
T5H23c;(▲) receptorsisolatedfrom cells infectedfor 30 minutes withT5am231. P/Po is the
number of PFU at the indicatedtime divided by the number of PFU at zero time (Dunn &
Duckworth 1977).
The T5H23c (for 30 minutes) reducedthe rate of adsorptionby 90%. After the
infectionof T5am231, the fullyactive receptorswere able to be isolated. As a result, it was

7. seenthat the inactivationof receptors doescome from attachment of the phage. Rather, the
inactivation requires the whole DNA molecule to be injected. Thus, it can be seenthat the
receptorsare inactivatedand cannot be isolatedfrom the infectedcells late inthe T5
infectious cycle. Also, bothnormal and T5-infectedcellsrelease somethingthat can
inactivate T5. However, infectedcells do not release moreof this substance thanuninfected
cells. Following these observations it was concludedthat is not likelythat the infection
causes the release of the phage receptors. Therefore, duringinfectionof Escherichia coli by
bacteriophage T5, the cell surface receptors for the phage were inactivated. Consequently,
they couldnot be isolatedfrom the infectedcells, but the mutant of T5 observeddid not
cause the inactivation.
In conclusion, it is known that the receptorfor T5 phage on Escherichiacoli is the
FhuA gene product and that some substance inactivates the phage at or before lysis. It is also
known that deletionof the TonB box (amino acid residues 7 to 11) onthe FhuA gene will
eliminate functionof the gene. Fixation of the corkto turn the barrel through a disulfide
bridge betweenresidues eliminatedferrichrome transport onthe FhuA gene product
structure. However, it is not known if the phage inactivator is T5 or some other substance
(Tadesse 2012).
Lastly, a study by Demeric and Fano in 1945 observedstrainB Escherichiacoli inthe
presence of coliphage T1, T2, T3, T4, T5, T6, and T7. Their goal was to examine multiple
resistance and identifyif multiple resistance transpiredfrom independent mutations. The
researchinvolved Escherichia coli that was already resistant to T-phages. Double mutants
were created(another experiment was conductedonthese resistant forms.) Results showed

8. that the patternof resistance was not different for single mutants incomparisonto double
mutants. They also discoveredthat the mutants resistant to T5 coliphage were also resistant
to T1 coliphage (Demerec & Fano 1945). Followingthe inconclusive results of these studies
in this new fieldof research, the two main objectives of this experiment are to generate
Escherichia coli that are resistant to T1 bacteriophage and to classifythe Escherichia coli
mutations that result followingresistanceto infection.
Materials and Methods
Preparation
Tryptic Soy Soft Agar (TSA) tubes and Tryptic Soy Broth(TSB) tubes were created
where a mixture of 6.75gof TSB in 225 mL deionizedwater. Similarly, a mixture of 3.6g
TSB and 1.2gagar in 120mL deionizedwater was microwaved until dissolvedand dispensed
to make 28 tubes of TSA soft agar. Escherichiacoli, strainB, was also inoculatedfor growth
overnight on a TSA streakplate.
Plaque Assay
Plaque assay was done to isolate Escherichiacoli mutants that are resistant to T1
infection. Ten-foldserial dilutions of T1 phage were performed to 10-7. Next, 0.1mL of fresh
brothculture Escherichia coli (strainB) was added to a sterile snap-capculture tube along
with 0.2mL of each dilutedphage, plated separately. The phage and Escherichiacoli were
added and the screw-captubes of TSA were then temperedina 46°C water bath before
adding 3mL of TSB. The screw-captube was inverted twice to mix the contents beforebeing
added to a soft agar tube. The soft agar tube was also inverted twice to mix the contents,

9. avoiding bubbles, and then immediatelypouredonto a Tryptic Soy agar base agar plate. This
plate was then swirledin a figure 8 patternto disperse the soft agar. It was left to solidifyat
room temperature before being invertedand placed into the 37°C incubator for 24 hours.
This procedure was attemptedtwice for this experiment.
Regrowth of Escherichiacoli Mutants
A freshTSA plate was made from Shaigan Bhatti’s colonies, previouslyverifiedon
EMB. The focus was on colonies that grewwell. A loopful of each numberedcolonywas
transferredfrom Shaigan’s TSA plate to a freshTSA plate (usingthe same numbering
system.) A small smear was made for colonynumbers 1, 4, 6, 7, 9, 12, 18 and 19. A loopful
of fresh Escherichiacoli stockculture was also added onto the TSA plate as a control. The
plate was incubated overnight at 37°C before beingstoredat 4°C. The plate was sub-cultured
onto a freshplate every few weeks.
MRVP Testing
MRVP (Methy Red Voges-Proskauer)testing was a vital confirmation test for
Escherichia coli necessaryto ensure no contaminationoccurred. The MRVP testingwas
conductedon Shaigan Bhatti’s individual colonies from researchconductedin2015. The
Voges-Proskauer portionof the test required -naphthol solution 40% KOH.
Genomic DNA Isolation
The final portion of this research is still inprogress. This is being accomplishedby
Polymerase ChainReaction(PCR) and amplification. The genomic DNA from positive
cultures of mutants has been isolated and will soonbe PCR amplified. FreshTSB broth

10. cultures were made from Shaigan’s cultures on the TSA plate for this procedure. Usinga
DNeasy Bloodand Tissue Kit, the DNA from colonynumbers 1, 7, 12, 18 and 19 have been
isolatedvia the followingprocedure: 1mL of freshbrothculture was transferredto a
microcentrifuge tube and centrifugedfor 3 minutes at 10,000 rpm. The supernatant liquid
was discardedand this stepwas repeated. The cells were re-suspendeduntil the pellet was no
longer visible and the DNeasy Bloodand Tissue Kit was usedfrom this stepon. Next, 20μL
of Proteinase K and 200μL of Buffer AL were added and mixed by vortexing. The tubes
were thenincubated for 20 minutes at 57°C using the temperature block. An addition of
20μL ethanol was mixed for 15 seconds byvortexing before the entire mixture was
transferredto aspin column. The spincolumn was then centrifugedat 8,000 rpm for 1
minute and the liquid was discarded. Before this stepwas repeated, 500μL of Buffer AW1
was added, without wetting the rim. The liquid was once again discardedand 500μL of
Buffer AW2 was added. The spin columnwas centrifugedat 13,200 rpm for 3 minutes and,
once the liquid was discarded, the columnwas transferredto a new microfuge tube with a
snap cap. An addition of 100μL of Buffer AE was left to incubate at room temperature for1
minute before beingcentrifugedat 10,000 rpm for 1 minute. Lastly, the spin columnwas
discardedand the genomic DNA was storedina freezer box. The next stepwill be PCR
amplification.
In conclusion, Figure 4 depicts the general steps taken in this researchfrom
combinationof Escherichiacoli brothand T1 coliphage to plaque assay to the confirmation

11. testing. In addition, the genomic DNA from confirmedmutants will be isolatedand analyzed
using PCR in the last few weeks of the semester.
Figure 4. The steps taken to combine Escherichia coli andphage, perform plaque assay, and
perform confirmationwithEMB and MRVP testing

12. Results
Plaque Assay
The results for the plaque assay were inconclusive. The procedure was conducted
twice but yieldedlittle to no plaques both times. However, there was a multitude of
Escherichia coli growthonthe plates. In the second attempt, the serial dilutions 10-1-10-3
were slightlylumpy and appeared to have some bubbles. Figure 5 displays the second
attempt at plaque assays followingserial dilutionof T1 coliphage. Since no plaques were
producedin the first attempt, no picture was taken.

13. Figure 5. Plaque assays of a) 10-1, b) 10-2, c) 10-3, d) 10-4, e) 10-5, f) 10-6, and g) 10-7 T1
phage serial dilutions
a) b) c)
d) e) f)
g)
Regrowth of Escherichia coli Mutants
The regrowthof Shaigan’s mutant colonies was successful as eachsmear showed
growth. Consequently, theywere used to go forthwith MRVP confirmationtestingto ensure
that the growth was Escherichia coli andnot a contamination.

14. MRVP Testing
The development of a pink to ruby redcolor inthe mixture, from 30 minutes
to 4 hours followingthe addition of the reagents was consideredpositive. The Methyl red
portionof the test required methyl redindicator. A distinct redcolor indicatedapositive test
and a distinct yellowcolor indicateda negative test. The methyl redtest demonstrated a
microorganism’s abilityto produce stable acidend products from the mixed-acid
fermentationof glucose. Acidproducedduring the fermentationlowers the pH of the
medium, resultingin a positive test with methyl red(positive=acid= red; negative=alkaline
or neutral = yellow). The Voges-Proskauer test was used to identifymicroorganismsthat
produce acetoinfrom glucose metabolism insteadof stable acids. Upon the addition of -
naphthol and KOH, the acetoin was oxidized to diacetyl which interacts withpeptone
components to yielda redcolor (positive reaction). No color change or a yellowish-orange
color was considerednegative.
The MRVP confirmationtest was successful. Eachsample exhibited positive results
for the VP portionof testing, thoughcolonynumbers 4, 7, and 12 were questionable as they
were onlyslightly pink. Colonynumbers 1, 18, and 19 showed the strongest positive results
with a deep redcolor. Results for the Methyl-redportionof the test were also all positive,
however colonynumbers 4, 7, and 12 were questionable once again with a slight pink color.
As seenbefore, colonynumbers 1, 18, and 19 showed the strongest results witha deep red
color.

15. Genomic DNA Isolation
Genomic DNA isolationfrom freshbrothcultures of Escherichiacoli were conducted
to analyze the DNA via PCR. This portionof the experiment is ongoingand has yet to be
determined.
Discussion
Turbid brothindicatedsuccessful growthafter inoculationof Escherichiacoli inthe
TSB. Growth was also confirmedonthe streakplate with cloudyyellow smears. The MRVP
test was conducted on the colonynumbers (1, 4, 6, 7, 9, 12, 18, and 19) that grew best on
EMB plates in Shaigan’s prior research. Results oneachcolonynumber were positive which
confirmedthat the mutants from Shaigan’s researchwere definitively Escherichiacoli and
not a contamination. Colonynumbers 1, 18 and 19 showed the strongest positive results with
a deepred color while colonynumbers 4, 7 and 12 showed the weakest results with a pale
pink color. As a result of these conclusions, the researchfocus shiftedto colonynumbers 1,
7, 12, 18 and 19.
In addition to lookingat Shaigan’s mutants from last semester, an attempt at growing
and isolating new T1 bacteriophage was made. However, the plaque assay portionof the
experiment was unsuccessful so no PFU/mL was concluded. Two attempts were conducted,
neither of which resultedin a significant amount of plaques. However, there was successful
growth of Escherichia coli onthese plates which was confirmedbythe fact that they were
tintedyellow with a turbid appearance. A possible error was the temperature of the water
bath for the soft agar as the first fewserial dilutions clumpedup as soonas they were

16. transferredto the final plates. However, Dr. Risleytestedthis and ruledout that possibility.
Some error could have also come from technique. WhenDr. Risleyrepeatedthe procedure
(for a thirdattempt) it went smoothly. Nevertheless, there was still not a significant number
of plaques. To fix this, more phage stockwould needto be orderedand the procedure would
need to be repeated. Consequently, the researchfocus shiftedtowardthe genomic DNA
isolationand analysis (still inprogress).
This PCR procedure was conductedon the mutants from colonynumbers 1, 7, 12, 18
and 19. The first attempt was unsuccessful as there was not enough cell growth in the fresh
TSB cultures. As a result, no pellet was createdinthe first step of centrifuging. In the second
attempt, the cells were further grown in TSB in the 37°C incubator overnight before
repeatingthe procedure. In this secondtrial, colonynumbers 18 and 19 had enough cells to
conduct the experiment (indicatedby a turbid broth). However, colonynumber 1 still didnot
possess enoughcell growth(indicatedby a clear broth) and thus no pellet formed. The
experiment was continuedwith colonynumbers 18 and 19. A third attempt at this procedure
was conductedon colonynumbers 1, 7, and 12 successfully.
In conclusion, this procedure is necessarybecause the DNA sequence canbe studied
and comparedto a DNA sequence of non-mutatedEscherichia coli allowingfor a deeper
understanding of these mutants and possible indicationof where to proceedwith farther
research.

17. Future Research
Further researchwill be done to produce more Escherichia coli resistant to T1
bacteriophage. A new phage stockneeds to be orderedto redo the plaque assay. Once the
genomic DNA has been analyzed, it can be comparedto the DNA of normal Escherichia coli
and farther genetic testingcanbe done in an attempt to eventually find how antibiotic
resistance canbe stoppedor at least maintained.
Acknowledgements
I would like to thank Dr. Risleywho advised me through this research. She has
become a role model and support system to me through many classes and ultimately, this
researchopportunity. Her guidance and patience contributedto aprime learning experience
in a settingI was initiallyuncomfortable with. Without her help and encouragement, this
researchwould not have beenpossible.
Additionally, I’d like to express mygratitude to the University of Mount Union
BiologyDepartment who suppliedthe resources andprior educationto make this research
possible.
Lastly I would like to thank Shaigan Bhatti for her prior researchthat allowedme to
conduct these experiments.

18. References
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