1. Bacillus anthracis Single Stranded Binding Protein: A Potential
Antibiotic Target.
Allie Trafton
Katelin Krenzke
7 December 2015
ABSTRACT: All organisms on Earth have their own Single Stranded Binding protein which protects single
stranded DNA and aids in DNA recombination, repair, and replication. SSB is a prime candidate for an antibiotic
target, especially in the case with Bacillusanthracis, the bacteria which causes the Anthraxinfections. The SSB
gene was cloned, then the SSB protein was induced, and the SSB protein was purified. Using EMSA, fluorescent
spectrometry, FRET analysis differences between Bacillus anthracis and E. coli SSB protein were found. The
largest difference was the lack of the (SSB)35 binding mode in Bacillus anthracis which led to the conclusion that
SSBBA may exist as a monomer rather than a tetramer like E. Coli SSB. This difference can be furthered study to
determine whether or not this can be a target for antibiotics.
INTRODUCTION
DNA is normally double stranded,butthe
strandswill unwind fromone anotherin particular
instances,suchas:recombination,repair,and
replication.However,single stranded DNA (ssDNA)is
prone to chemicaland nuclease attacks that damageto
the DNA that cannotbe easily fixed1
. But thanks to the
Single Stranded Binding protein (SSB),the ssDNA is
protectedfromsuch attacked.SSBs can also recruit
different genome maintenance proteinsand take themto
theirsites ofaction2
.Single Stranded Binding proteins
are essentialforlife to proceed as it has thusfar. SSBs
are made up of fouridenticalsubunitsthateach has an
acidic tail to which the SSB protein usesto recruit other
proteins.SSBis able to bind in two different modes,
(SSB)35 and (SSB)65. The 35 binding mode occurs in
low salt conditionsandthe ssDNA bindsto multiple
SSB tetramers.The 65 binding mode occursin high salt
conditionsandthe ssDNA wrapsitselfaround only one
tetramer.
All life on this earth have some sortofSSB
within theirbeing,including bacteria suchas Bacillus
anthracis.Bacillus anthracis is a large,grampositive
bacteria3
which is able to produce heat resistant spores4
.
These spores are commonly found in the ground,which
explains why grazing herbivores are the most likely to
consume the spores.Once the spores enteredan
organism,they can infect the organismwith anthrax.
When humans get infected with anthrax,it typically
occurs when the individuals handle orcome in contact
with sickanimals3
. There are three different types of
anthrax: cutaneous,pulmonary,andgastrointestinal.
Pulmonary anthraxis what most people imagine when
they hearthe word anthrax.Pulmonary anthraxis used
in bioterrorismattacks,such as the attacks that took
place in late 20013
.
There are vaccines foranthrax,but they aren’t
available to the generalpublic.However,if we are able
to find some unique propertyofthe SSBprotein of
Bacillus anthracis,a property that differs fromthe
highly studiedE.coli SSBprotein,we can use it as an
antibiotic target.
METHODS
Transformationofplasmids and expressiontest
Initially, the Bacillus anthracis SSBgene was contained
within a pUC57 plasmid, which lacks a T7 promotor.
The SSB gene thus neededto be ligated fromthe pUC57
plasmid and into a pET28a plasmid which contains the
promotor.A restriction digest tookplace usingNdeland
BamHI restriction enzymes to cut the SSBgene from
the pUC plasmid and to open the pET28a plasmid.An
agarose gelwas run to ensure theSSBgene was cutFigure 1: The Single StrandedBinding
Proteinis a tetramer with 4 acidic tails
which recruits other proteins.

2. from the pUC57 plasmid and the pET28a vectorwas
opened.The SSBgene and the pET28a vectorwere then
ligated together. Afterthe two were ligated with one
another,DH5αcells and the ligation mixture were
mixed together,placed on ice,heat shocked,andthen
placed backon ice.Once the mixture cooled,it was
mixed with LB-media and placed on a shaking incubator
for an hourand then the media was plated.The
following day,one colony was picked andplaced in LB-
media and incubatedovernight.Thesecells were then
spun down. The purified plasmid DNA was isolated
using the Qiagen MiniprepKit.A restriction digest
followed by gel electrophoresis tookplace to determine
if the SSB gene was inserted intothe plasmid.
Purificationand IsolationofProtein
To activate the expression ofthe SSBgene,BL21-DE3
cells were transformed with the pETa-SSBplasmid. The
BL21-DE3 cells carry the inducible gene forT7RNA
polymerase which will formthe SSB protein.A BL21-
DE3 colony was grown overnightin LB-kan media and
induced using IPTG.The induced culture was then
titrated with PEI and spun down untilthere was
separationofthe supernatantand pellet.The PEIpellet,
which contained the SSBprotein,was resuspended with
bufferT0.4
and was mixed with ammonia sulfate.The
ammonia sulfate salts outthe protein fromsolution,
which leaves the purified protein in the ammonia sulfate
pellet.
Testing BindingAffinity ofSSB Protein
To determine whetherornot the purified SSB protein
would bind to single strandedDNA,an Electrophoretic
Mobility Shift Assay(EMSA)was performed.First the
concentrationofthe purified protein was measuredand
then a series ofreactionscontainingvarying
concentrationsofthe SSBprotein,10x buffer,10µM
dT70 DNA, and waterwere made. A 30 µL sample of
each reaction was thenloadedinto a 10%
polyacrylamide gelmade with TAE bufferand ran at
100 volts.The gelwas then stained andan image ofit
was taken with the stormimager.
Testing SSB Kinetics
The rate or speed at which the Bacillusanthracis SSB
protein bound to ssDNAat varying SSBconcentrations
was examined. In 6 different centrifuge tubes,6
different reactions containingvaryingamountsof2xT
buffer,SSB protein,and waterwere made. In a plastic
culture tube,2xbuffer, 135 µM 5’ Cy3 (dT)64 ssDNA,
and deionized waterwere mixed together.Then the
binding ofthe protein sample and DNA sample were
measured usingfluorescent spectroscopy.
Using FRET analysis to checkSSB bindingmodes
Lastly,FRET was performed to determine which modes
the Bacillus anthracis SSBprotein appearedin when the
protein was bound to theDNA in both high and lowsalt
conditions.Forboth highsalt and lowsalt conditions,a
400 nM concentrationofthe SSBprotein and a 20 nM
concentrationofthe stockDNA was used.Forthe low
salt assaythe SSBprotein,bufferT0.02
,and deionized
waterwere mixed in one reaction tubeand stockDNA,
bufferT0.02
, and deionized waterwere mixed in another.
For the high salt assay,the same reactions were made
but bufferT0.2
was used in place ofbufferT0.02
. Afterthe
reactions were made,the respectedDNA and protein
reactions were mixed and excited with a 515 nm
wavelength.
RESULTS
Purificationand IsolationofProtein
The gel in figure 2 contains allofthe reactions that have
been performed in orderto achieve a purified SSB
protein.Once the lac operon had been induced with
IPTG, formation ofthe SSB protein was initiated as
seen in well 3 of the figure. Afterboth the PEI
precipitation and theammonia sulfate precipitation,the
SSB protein was foundin the pellet.The final purified
SSB protein was locatedin the ammonia sulfate pellet.
Figure 2: Purificationgel of the Bacillus anthracis
SSB protein.The ammonia sulfate pelleton the far
rightcontains the pure SSB protein.
Testing BindingAffinity ofSSB Protein
The ODs of the purified protein sample were taken at
280 nm and 345 nm. Using thesetwo values and the
extinction coefficient ofthe Bacillus anthracis SSB
protein,the concentrationofthe SSBprotein in the
sample was determined,all of which is presentin table
1.

3. OD280 0.417
OD345 0.031
Extinction Coefficient 11,460
[SSB] 84.2 µM
Table 1: Data needed to determine the concentration
of SSB proteinin sample.
Afterthe gelwas run,there was one visible band present
in each ofthe wells. As shownin figure 3, as the
concentrationofthe protein increasesthe lessunbound
DNA there is present.The amount ofthe unbound DNA
decreasesasthe concentrationofthe protein increases.
It appears that evenat a lowconcentrationofSSB
protein,0.01 µM to be exact, most if not all of the
unbound DNA is bound to theSSBprotein.This
indicates thatthe Bacillusanthracis can bindto the
ssDNA.
Figure 3: The top rows of boxes contain the protein
bound to DNA. The bottom boxes contain the
unbound DNA.
Testing SSB Kinetics
When the DNA reaction andthe protein reactions were
mixed with one another,fluorescence spectrometry
indicated that theprotein did in fact bind to the DNA as
expected,as seen in figure 4. As the concentration ofthe
SSB protein increased,the amount ofCy3fluorescence
increased as well.
To determine the binding constant ofthe Bacillus
anthracis SSBprotein,the initialrates ofeach
concentrationwere plottedagainsttheirrespective
concentrations as seenin figure 5. Then the slopeofthe
line was determined,which is the binding constant of
the protein and in this case it was 51.894 µM-1
s-1
.
Figure 5: The rates of the reactions plotted against
their respective SSB concentration.The slope of this
line was determined to be the binding constant.
Using FRET analysis to checkSSB bindingmodes
When the lowsalt analysis tookplace,high FRET was
seen.This indicatedthatthe binding mode the protein
was in made it so that the Cy3and Cy5were nearone
another.In the high salt analysis,lowFRET was seen
indicating the two ends of the DNA were not nearone
anotherwhich enabled the Cy3end to excite the Cy5
end ofthe ssDNA.When this experiment was
previously donewith a lower protein concentration,high
FRET was seen in the high salt analysis as it was seenin
the low salt analysis.
Figure 6: FRET analysis of the Bacillus anthracis
SSB proteinin both highand low saltconditions.
y = 51.894x + 62.311
0
50
100
150
200
0 0.5 1 1.5 2 2.5
Rate(s-1)
[SSB] (µM)
Figure 4: The fluorescence ofCy3 at differing
concentrations of SSB protein.

4. Figure 7: The alignmentof the amino acid sequences of E. col and Bacillus anthracis SSB protein
DISCUSSION
The main goalof this research was to
determine whetherornot there were any significant
differences between theSSBprotein of E. coli and
Bacillusanthracis that could be used asa potential
antibiotic target.When theamino acid sequences ofE.
coli and Bacillusanthracis were compared to one
another,it became evident that there were goingto be
differences in howthe Bacillusanthracis SSBprotein
interacts with ssDNAcomparedto the SSBprotein of E.
coli.All ofthe tryptophanamino acids in the E.coli
SSB protein amino acid sequence were replacedwith
otheraromatic amino acids in the Bacillusanthracis
protein.In the E. coli SSB protein,the tryptophan in the
54th
position playsa role in stabilizing the protein-
ssDNA complex5
.But in the Bacillusanthracis SSB
protein the amino acid that is located in this location is
phenylalanine.Whetherornot this change in amino acid
affected ssDNAbindingwill be discussedshortly.
Anotherkey difference betweenthe two sequences can
be seen in the acidic tail region of the sequence,which
is the last 6 amino acids in the sequence.This regionis
needed forinteractionswith proteinsthat dealwith
recombination,replication,and repair6
.In the E. Coli
sequence,there is an isoleucine that is replacedwith a
leucine in the Bacillusanthracis sequence.Although
both amino acids share characteristics,this small
difference could potentially lead to a target for
antibiotics.
Once EMSA was performed,it was clearly
evident that SSBBA can bind to ssDNAjust like SSBEC.
At a low concentration ofthe SSBBA,most of the DNA
in the reaction was boundto the SSBprotein,there was
very little separationofthe DNA and the protein seenon
the gel.There could have been two reasons forthis:that
there just wasn’t enoughtime given to run the gelor that
the concentration ofthe initialprotein sample was
incorrect.Looking at the gelin figure 2, it appears as the
protein concentrationwas off,meaning the actual
concentrationofthe protein sample was much higher
than was initially calculated.
Using Cy3 fluorescenttag,which fluoresced
when the SSBBA protein was bound to the DNA,the
binding constantofthe SSBBA was able to be
determined.The calculated binding constant ofthe
Bacillus anthracis SSBprotein was 51.894 µM-1
s-1
.The
binding constantofthe E.coli single strandedbinding
protein is 582 µM-1
s-17
.This indicates that thebinding
constantofthe Bacillus anthracis SSBprotein is much
higherthan the E.coli SSBprotein,which means SSBBA
is more likely to bind to DNA than that ofthe SSBEC.
However,the calculated binding constantfound in this
protocolmight have beenincorrect since thedata
gathered did not create a linearline itself, a v shaped
line was seen instead.In future research,smaller
concentrations ofthe SSBprotein would have beenused
to ensure a more accurate binding constant.
The area where the largest amount of
difference between the two SSBproteins was found
using FRET analysis to studythe binding modesofthe
proteins.Once the FRET analysis tookplace,it
appeared that SSBBA only had one bindingmode.In
both the high and lowsalt conditions,high FRET was
seen.This means thatthe Cy3end ofthe ssDNA was
nearenoughthe Cy5

5. Figure 8: The binding modes of E. coli SSB protein.
end to excite it and for this to occur,the protein needs to
be in the (SSB)65 binding mode so that theDNA
completely wraps aroundone tetramer. Bacillus
anthracis is not able to forma stable tetramer,but rather
it has been hypothesized that it has a transienttetramer6
.
This means that the fourmonomers ofthe protein are
unbound fromone anotherandonly come togetheronce
bound to the ssDNA.Going off of this theory,the
(SSB)35 binding mode would exist but ratherthan a
numberof tetramers binding to thessDNA it would be
the individualmonomers that would bind to the ssDNA.
Since only high FRET was seen in the experiment it can
be assumed that there isn’t an (SSB)35 binding mode for
the SSBBA or if there is one then the (SSB)35 complex
concentrationwas sosmallthat it was just not seen6
.
The greatestamount ofdifference between E.
coli and Bacillus anthracis SSBprotein is the structure
of the protein itself.There are differences in the amino
acid sequencesbetweenthe two bacteria which affects
hydrogenbond formation6
.While there is a stable
tetramerfor E. coli SSB, the amino acid differences in
Bacillusanthracis preventsformation ofthe tetramer.
So the SSBBA exists as monomers that eitherthe DNA
can wrap completely around to showhigh FRET results
in all instancesorthat the monomers come togetheras a
transient tetrameronce boundto ssDNA.There is even a
small difference between the amino acid sequences of
the acidic tail of the protein,where there is an isoleucine
in E. coli and in Bacillusanthracis there is a leucine in
that same position.In the future,these two differences
can be studied more intensely to determine if either
could lead to a potentialtarget forantibiotics.
ACKNOWLEDGEMENTS
I would like to thankDr. Edwin Antony andJessica
Andersonforproviding instructionand help overthe
duration ofthis research.
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