Evolution of a ‘Recalcitrant’ form of the Bacteriophage ST-1

Preston Stolte

BIO 377
Heineman
Fall 2010
INTRODUCTION

When studying the ability of bacteriophage to adapt to new hosts, it is important to study

how the environment affects the phage’s ability to infect its host and create viable progeny. Most

phage undergo the lytic cycle, however, some phage remain in the lysogenic cycle until

environmental conditions favor lysis. For either of these cycles to occur however, a phage must

first attach to a host cell and insert its genetic information; because of this, it is important to

study how environmental factors may affect a phage’s ability to adsorb to the host cell. The

bacteriophage ST-1 is particularly useful in studying this phenomenon; due to its ability to

convert between active (infective) and inactive (non-infective) structures in response to

temperature, we are able to study the effect of environment on adsorption rates and phenotype.

ST-1 is a small single-stranded DNA (ssDNA) virus that is related to the bacteriophage

ΦX174 (1). ΦX174 is a bacteriophage that infects Escherichia coli C and can convert between

two forms, Φ and Φ*. The Φ* form is inactive at 4°C and more resistant to extreme temperatures

(65°C); the Φ form, is able to infect host cells but is sensitive to heat (2). Φ*X174 changes into

the more heat sensitive form ΦX174 at temperatures around 37°C while ΦX174 will convert

back into the heat resistant form at 4°C (3).

ST-1 bacteriophage differs from the ΦX174 bacteriophage in a few ways that make the

virus more advantageous for observing the effects of environment on viral evolution. One of the

more important things to note is that ST-1 bacteriphages have a more limited host range because

unlike ΦX174, which can infect K-12 and C strains of E. coli, ST-1 bacteriophages can only

infect K-12 strains of E. coli; this makes ST-1 more beneficial for research because the functions

of host cell systems of K-12 strains are better understood than those of C strains of E. coli (1, 4).
It has also been shown that ST-1 only requires the dnaG and dnaE gene for DNA replication,

which means it uses fewer host proteins and follows a more simple replication process than

ΦX174 (1). The simplicity and specificity of the ST-1 bacteriophage make it an ideal

bacteriophage for viral evolution studies.

The goal of this project is to encourage the evolution of the ST-1 virus into a

‘recalcitrant’ phage, or a phage which takes significantly longer to convert from the inactive

(Φ*) form to the active (Φ) form at 37°C. This is accomplished by modifying temperature and

growth conditions, such as preventing lysis, to select for a recalcitrant ST-1 phage that remains

in the inactive form. Recalcitrant phage are selected for by growing a lysate in the presence of E.

coli Δrep cells which are a form of host cell that does not allow for the creation of ΦX174

progeny. The production of ΦX174 is prevented by Δrep cells because the viral DNA is allowed

to enter the cell and is incorporated with the host cell DNA, but the parental RF DNA strand is

then not replicated meaning that no new phages are produced (5). Those ST-1 phage which

remain in the inactive form will not have been selected against by the Δrep host cells and

presumably will have evolved a recalcitrance to activation. This will be discussed in more

thorough detail in the methods section.

If we are able to obtain a phage which actually shows ‘recalcitrance’ we will be able to

study phenotypic plasticity in phage using ST-1 as a model. Phenotypic plasticity is the ability

for a genotype to display different phenotypes in response to changes in environmental

conditions (6). Not only will this provide us the ability to study phenotypic plasticity but we will

also be able to study processes of evolution in individuals with a plastic phenotype. For example

we will be able to see whether plasticity increases as a response to selection (6) or how difficult

it is to adapt and organism with a plastic genome to become less plastic. We will also be able to
see if the loss of plasticity in the recalcitrant phenotype is favored under different environmental

conditions.

METHODS

To begin this project an active lysate of the ST-1 phage was made from the stock of wild-

type ST-1 phage provided by Dr. Ian Molineux. The lysate was grown in 10 mL of luria broth

(LB) using 75 ul of -lac cells allowed to grow in a shaking water bath set to 37 °C and 200 rpm

for one hour. It is important to note that when working with ST-1 bacteriophage, a divalent

cation such as Ca++ must be present for adsorption and eclipse to occur. This can be

accomplished by adding aqueous CaCl2 to the growth medium (5mM/10mL). After the host cells

had grown for one hour, 100 ul of ST-1 phage stock was added to the flask and allowed to grow

for 3+ hours until the flask lysed. This active ST-1 lysate was tittered (4E7 phage/mL) and saved

in a conical vial and used to prepare an inactive lysate for later use.

It is important to note that the titer of the initial ST-1 lysate decreased over time until no

phage was present. This may be due to the phage becoming trapped within the cell debris at the

bottom of the conical vial or it could be to the death/disintegration of the phage. Talking with

other lab members who have worked with ST-1 indicates that this is a common problem.

Because of this, an active lysate had to be grown for a second time in early October (10/8/10).

However, before the active lysate was grown, a 1mL lysate was grown by growing 45 ul of -lac

cells for one hour, adding 10 ul of ST-1 phage stock and allowing the lysate to grow overnight in

the presence of the mutagen N-methyl-N-nitro-N-nitrosoguanidine (5ug/mL). The reason for

growing a lysate in the presence of a mutagen was to increase the genetic diversity in the ST-1

lysate to hopefully encourage more diversity in mutations and adaptations. The titer of the
mutated lysate was 1E8 phage/mL, and an active lysate was grown using 10 ul of this mutated

lysate which had a titer of 4E10 phage/mL.

After the active lysate is made, it is used to make an inactive lysate (a lysate of ST-1

phage in the inactive ST-1* form). This means that the active ST-1 phage are allowed to infect

the –lac cells, but before the lytic cycle is allowed to be completed lysis is prevented by the

addition of sucrose (7). This was initially done by growing 45ul of -lac cells in a 10 mL solution

of LB with 20% sucrose (w/v) and 5mM CaCl2. After the -lac cells have grown for an hour, 250

ul of the activated ST-1 lysate was added to the flask and allowed to grow for 1.5 hours. The

addition of the sucrose is used to prevent lysis of the -lac cells after infection (5). A sample of the

solution is then plated to ensure that lysis has not occurred. We then cool the flask to ensure that

all phage are converted to the inactive ST-1* form and use chloroform to lyse the cells and

lysozyme (2mg/mL) to rupture the cell walls thus allowing the ST-1* to escape the host cell

while remaining in an inactive form. This inactive lysate is then collected in a conical tube and

tittered.

Using the above procedure, lysis of the -lac cells still occurred meaning that the sucrose

was not properly preventing lysis. Since the sucrose solution was autoclaved, I looked at the

effects of autoclaving a sucrose solution and found that when autoclaved some of the sucrose

will be converted to glucose. Because of this a new sucrose solution was made which was filter

sterilized instead of autoclaved and the inactive lysate was grown again; however, using the new

sucrose solution only provided marginally better results. I then looked at the protocol that

Danielle Rodriguez followed and saw that she added dry sucrose to the solution so I tried

growing a lysate using 6.348 grams of dry sucrose. Dry sucrose can be autoclaved for up to 12

minutes without melting, forming a solid, or converting to glucose. Adding dry sucrose to the
lysate around 10 minutes after the addition of active ST-1 worked substantially better and

effectively prevented lysis.

After the inactive lysate was made, attempts were made to adapt the phage to be less

inclined to convert from the inactive ST-1* form to the active ST-1 form at 37 °C. This was done

by preparing four separate flasks using 45 ul of Δrep cells grown in 10 mL of LB with CaCl2.

We then warmed samples of the inactive lysate for different intervals of time (30 minutes, 10

minutes, 2 minutes, and .5 minutes) to initiate transformation from the inactive to the active

state. These warmed samples were then added to the flasks of Δrep cells and transferred to a 26

degree Celsius water bath where they were passaged for 1.5 hours. The purpose of transferring

the flasks to the 26 degree water bath is to prevent the phage samples from further converting to

the active form. After numerous failed trials, I eventually realized that the reason all of the ST-1*

phage were being converted to ST-1 despite the amount of time spent warming the sample was

due to the transfer of a warm 37 degree flask to the 26 degree water bath after the phage had

been added. The protocol was modified by transferring the flasks containing Δrep cells to the 26

degree water bath ten minutes before the inactive lysate samples were added to ensure that the

flask temperature was also 26 °C to prevent further conversion.

The next step in the protocol is to titer the time trial passages using –lac cells, the

formation of plaques on a plate would indicate that ST-1* phage had not converted to the active

form indicating the emergence of a slight recalcitrance. We would then use this sample to grow a

new inactive lysate and we would then repeat the process over again.

RESULTS AND DISCUSSION

 While there has been a lack of significant results in terms of phage adaption, there have

been numerous results which have led to numerous modifications and increased efficiency of the

experiment protocol. One of the first and most frustrating results was the inability of the 80%

(w/v) sucrose solution to prevent lysis of the –lac cells by ST-1. Due to this I first looked at the

effects of autoclaving sucrose solutions, and as mentioned in the methods section autoclaving

sucrose causes some of the sucrose molecules to decompose into glucose which would not

prevent lysis. However, after attempting to make an inactive lysate using an 80% (w/v) sucrose

solution which was filter sterilized I realized that the problem was not autoclaving the sucrose.

The filtered sucrose solution performed slightly better than the autoclaved sucrose solution but

finding the titer samples taken from a passage using the filtered sucrose solution at 15, 30, and

60 minutes revealed that lysis was in fact still occurring because the titer of the phage was

continually increasing.

After this test I looked at Danielle Rodriguez’s results to see what she did to prevent lysis

and she sprinkled 6.348 grams of dry sucrose into the flask 10 minutes after ST-1 infection to

give a 20% (w/v) solution. I then autoclaved some dry sucrose and then grew two inactive

lysates to test the effectiveness of autoclaved dry sucrose. One lysate was grown using the

filtered sucrose solution added along with the LB; in the second lysate, lysis was prevented by

adding dry sucrose 10 minutes after infection. After tittering the results it was clear that the dry

sucrose was significantly more effective at preventing lysis then either of the sucrose solutions.

The titer (before artificial lysis) of the inactive lysate using the filtered sucrose solution was 2E9

phage/mL while the titer of the lysate made using dry sucrose was 2.3E6 phage/mL. Ideally the

titer of the unlysed lysate should be 1E6 phage/mL because that is the expected number of phage

which were initially added to the lysate. A titier of 2.3E6 phage/mL indicates that some lysis
may have occurred during the 10 minute window before sucrose was added, but that once

sucrose was added, lysis was effectively prevented.

While I do not definitely know why the sucrose solution failed to prevent lysis, some

possible reasons for its ineffectiveness include the sucrose dissolving in the LB and forming

glucose, or it is possible that the –lac cells absorb the sucrose and use it as a source of

nutrition/energy during their growth causing a reduction of the sucrose in solution. This second

hypothesis may explain why it is better to wait until after infection of the –lac cells by active ST-

1 bacteriophage because it would limit the amount of time that –lac cells could degrade the

sucrose. The reason that sucrose is added 10 minutes after infection is because it takes about 10

minutes from the moment of infection for ST-1 to lyse its host.

After an actual inactive lysate of ST-1* was obtained, passages were performed which

were designed to select against ST-1* phage which readily converted into the active form at 37

°C. The first few attempts at passaging ST-1* after warming the samples for various amounts of

time resulted in the formation of zero plaques when the results were titered. At first I thought this

may have been due to an error in plating or passaging technique because it seemed unlikely that

all of the ST-1* would be able to convert to the active form with only 30 seconds of warming;

however, after two more attempts it was clear that the error was not due to poor technique.

To check for the possibility that all of the inactive phage were converting to the active

form in less than 30 seconds of warming, another series of passages were performed where the

samples were warmed for 30 seconds, 15 seconds, 5 seconds, and 0 seconds. When these results

were titered, there were also no plaques for any of the samples, including the sample which was
just the lysate. This indicated a new problem which was that the titer of the lysate decreases over

time.

Some of the possible reasons for the decrease in lysate titer include the phage becoming

trapped in the cell debris and the phage simply degrading over time. In order to account for the

possibility that the phages were becoming trapped in cell debris, the new lysate that had to be

created was filtered using a syringe and a filter tip. The filtered lysate was then titered to ensure

that the phage were not filtered out along with the debris. The results of the titer indicated that

the phage were not caught in the filter so the filtered inactive lysate was then used for the new

series of warmed passages.

The second series of passages which attempted to select for a recalcitrant phage followed

the same protocol as the first set of passages with the exception of using a filtered inactive lysate.

However, the first few series of passages were ruined by personal errors in the passaging process

(wrong host cells, forgetting to transfer flasks to the 26 degree Celsius water bath, etc.). Finally, I

performed a passage without messing up the passaging protocol. When I titered the samples from

this series of passages, I also plated an undiluted sample of the inactive lysate as a control to

ensure that the lysate had not decreased in titer again. When I checked the plates there were a lot

of plaques on the section where the inactive lysate was plated indicating that the lysate had not

decreased in titer since it was made. However, there still were no plaques on any of the other

samples indicating that something must still be wrong.

This time the only explanation for the lack of plaques is that all of the ST-1* phage were

converted to the active form and then eliminated by the Δrep cells. Initially I was going to

attempt another set of passages using warming intervals of less than 30 seconds. Before I could
do this, I realized that a more plausible scenario for why all of the ST-1* were being converted in

every sample was because the flasks were being transferred from the 37 degree Celsius water

bath to the 26 degree bath immediately before the addition of ST-1* meaning the LB was still 37

°C when phage were added. This meant that the ST-1* were added to a 37 degree flask where

they were still able to convert to the active form for about 10 extra minutes before the LB cooled

to 26 °C and at this point all of the ST-1* would have been converted into ST-1.

The final modification to the passaging protocol was to allow the flask containing Δrep

cells to be placed in the 26 degree water bath 10 minutes prior to the warmed ST-1* lysate being

added. After passages were completed using this protocol, they were titered and there were

plaques present on both the undiluted sample that had been warmed for 2 minutes (2 plaques)

and the undiluted sample that had been warmed for 30 seconds (3 plaques). This not only

indicates that the problem with the warmed passages had been that they were still able to convert

to the active form when the LB in the flasks was not allowed to cool to 26 °C, but it also

provides a successful result to use when moving forward with this project. The results from this

set of passages are located in the Viral Evolution Freezer in a box labeled ST-1 Recalcitrant

Phages, Stolte, Box (1).

CONCLUSION

After finally obtaining a successful result from the warming step, the next course of

action is to take the phage which survived the longest heating (in this case the results from the 2

minute warming). Using the sample from this passage, a high-titer active lysate will be grown

and then this will be used to create another inactive lysate using the addition of sucrose. After

this the inactive lysate will be used for another set of passages which are warmed for various
times. While this project had many failures and a few modifications to the protocol, successful

results have been obtained and further passaging is now needed to determine if it is possible for

ST-1 to become less likely to transform from the inactive form to the active form at 37 °C.
References:

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Adsorption Behavior.” Virology. Vol. 34, No. 2 (February, 1968) pg. 366-370
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ΦX174 and Two of its Mutants.” Virology. Vol. 36, No. 3 (November, 1968) pg. 343-355
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