Competition between Escherichia coli and

Saccharomyces cerevisiae under resource limiting

conditions

Allen Hoffmann
Abstract: The creation of any bioreactor system is subject to the risk of contamination by
undesirable strains. This experiment simulates a contamination scenario by culturing
S.cerevisiae with E.coli in liquid cultures and agar plates over a two week period. When
brought into direct contact, E.coli grew faster than on open media. Unusual expression of
green fluorescent protein by E.coli was witnessed during this study. These findings indicate
a need to protect yeast cultures from E.coli contamination.
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Introduction:
The utilization of Escheria coli and Saccharomyces cerevisiae strains in biotechnological
applications is well documented. The creation of bio-reactor systems allows for the large scale
cultivation of microbial populations. Genetic engineering has allowed these microbial
populations to produce compounds of industrial or medical use. For all reactor systems
contamination by other strains is seen as a primary obstacle requiring strict aseptic protocols.
This paper describes research into competition between E. coli and S.cerevisiae cultured together
in resource limiting conditions as a means of simulating E.coli contamination of a yeast reactor
system.
There is a need to utilize easily identifiable strains for this experiment due to the lack of
advanced tools like florescence microscopy. The creation of plasmids expression florescent
proteins allows for visual identification of the strains being used. The E.coli strain HB101 K-12
is offered by Bio-Rad for use with the pGLO plasmid in transformation kits. The pGLO plasmid
from Bio-Rad, widely used in college biology labs, allows for the selective expression of green
fluorescent protein (GFP) when the cells are exposed to the sugar L-arabinose in the media. This
combination allows for an easy transformation and quick visual identification.
The growth of E.coli on luria-bertani (LB) media produces significant predictable results.
The LB media however contains no fermentable sugars indicating a need to supplement the
media with dextrose in order to facilitate yeast growth (Guennadi Sezonov, Danile Joseleau-
Petit and Richard D'Ari, 2007). In order to generate a fair competition the media both strains are
being cultured on must allow for growth of both species.
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A version of the mcherry plasmid coding for red fluorescent protein (RFP) was modified
to favor AT rich codons creating the yeast-enhanced mRFP plasmid. Yeast cells carrying this
plasmid produce a purple phenotype due to overexpression of the gene. This plasmid allows for
visual identification of fungal colonies and quick determination of plasmid carrying members via
fluorescence microscopy (Sabine Keppler-Ross, Christine Noffz and Neta Dean 2008). This
strains plasmid expression creates a striking visual difference from the E.coli to be used in the
experiment.
Contamination of a high density culture of S.cerevisiae by E.coli HB101 (Biorad)
inhibited the growth rate of a high density system. Strains able to form flocculent clumps were
more tolerant of contamination. Another implication of this study is that cells facing competition
may drop plasmids that slow down cellular replication (Luclia Domingues, Nelson Lima, Jose
A. Teixeira 2009). It can be inferred from this study that a sudden drop in plasmid expression
would be an indication of those yeast cells undergoing competition for resources.
Saccharomyces cerevisiae is one of the few yeast strains capable of anoxic fermentation
of sugars (Cornelius Verduyn, et al. 1989). This may indicate the possibility of resource
partitioning within deep liquid cultures where we would expect to see yeast surviving at the
bottom of the container in a region where E.coli growth is unsupported. When combined with the
issue of yeast flocculation it may be possible for liquid reactors to significantly limit the growth
of E.coli. An issue to look at in this experiment is how yeast handles contamination in aerobic
environments.

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Methods:
The E.coli strain HB101 K12 (Biorad) was on hand for the biology lab and was
previously transformed with the PGLO plasmid.
The S.cerevisiae strain was acquired from Dr. Neta Dean, Stonybook, NY, it was
provided previously transformed with the YemRFP plasmid as described in literature review.
Primary cultures were maintained by Dr. Mary Campbell, curator of the biological department
for Deanza Community College. Subcultures were maintained in liquid media and on agar plates
at 37C. The experiment was overseen by Dr. Brian McCauley in the molecular biology
laboratory at Deanza Community College.
Initial growth tests indicated a need to create specialized media for this experiment.
Standard LB broth was supplemented with dextrose while the sabouraud dextrose media was
supplemented with bacto-tryptone. The media was created according to manufacturers
directions and autoclaved before the sterile-filtered arabinose was added. The plates were hand
poured and incubated at 37C for 48 hours before use to insure they were free of contamination.
For each agar plate, 100 l of inoculated liquid media for each strain was transferred to
the agar plate via pipette then spread with an inoculation loop to cover half of the plate without
allowing the liquid to touch the walls of the plate or cross over the center. The plates were stored
in the incubator at 37C. Periodically the plates were removed from the incubator, placed into a
biological safety cabinet with an ultraviolet light source, and photographed using a canon digital
camera. Photographic sets were assembled for each of the plates described in the results section.
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For liquid cultures, 100l of inoculated liquid media from each liquid subculture were
added to 3.8ml of LB media or SAB media respectively and cultured in a shaking table incubator
at 37C. These hybrid cultures were then subjected to serial dilution then plated onto prepared
agar plates for observation.
Results:
Plate Media Strain Growth
(E./S.)
Fluorescence
(color)
Notes
1 LBmix1 E.coli + - GFP did not express as expected
2 LBmix1 S.cerevisiae + + red Yeast grows well on LB +dextrose
3 LBmix1 Both +E +S +red *grn Contact induced GFP expression
4 LBmix1 Both +E +S +red *grn Plate contaminated (mold) discard
5 SABmix1 E.coli Min - Expected GFP expression, min growth
6 SABmix1 S.cerevisiae + + red Yeast grows well
7 SABmix1 Both **/+ +red *grn E.coli rapid takeover, induced GFP
expression on contact, robust growth
8 SABmix1 Both **/+ +red *grn E.coli rapid growth where yeast
accidentally crossed over during
inoculation, rapid GFP expression
9 LBmixrnd2 Serial Dilution
of hybrid culture
+++/+ +red initial,
rapid green
flush
Rapid expression of GFP
10 LBmixrnd2 Serial Dilution
of hybrid culture
++/+ +red *grn GFP expression 1
st
seen between 3
yeast colonies, photo w/ dissection
scope
11 SABmixrnd2 Both +/+ +red *grn Weak initial E.coli growth,
accelerated on contact with yeast,
induced GFP on contact
12-16 The remaining plates were used to maintain subcultures throughout the experiment
Table 1: Agar plate trial results
The control plates for E.coli failed to demonstrate any expression of the pGLO plasmid.
No green florescence was observed over the life of the control plates. This indicates an
insufficient concentration of L-arabinose in the prepared mixture that was added to the agar
media.
The control plates for S.cerevisiae displayed a purple to pink phenotype and bright pink
fluorescence under UV illumination which corresponded to its documented behavior.
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In the aerobic environment of the incubator, E.coli demonstrated an ability to take over
the space occupied by the S.cerevisiae lawn of cells but the speed of takeover was specific to the
scenario created on the plate therefore two of the plates have been selected as visual references.
Plate 3 (LB mix1) was inoculated according to methods with both strains and allowed to
culture in the incubator; this was considered a successful inoculation due to the distinct visible
gap between the two lawns. Initially the S.cerevisiae lawn developed faster than the E.coli lawn
looking visibly thicker. Four days into the trial a white edge along the S.cerevisiae lawn could be
seen indicating presence of uracil and a dropping of the plasmid from the cells. Six days into the
trial a thin green line of florescence appeared along the boundary between the two strains
indicating activation of the pGLO plasmid. Following the 6
th
day robust E.coli growth is seen
along the boundary with florescence spreading backwards through the E.coli lawn. While
displaying aggressive growth, the E.coli did not significantly penetrate the yeast lawn.
Plate 7 (sab mix1) was inoculated according to methods with both strains and allowed to
incubate; this inoculation was seen as unsuccessful due to crossing over of the two strains as the
liquid settled into the agar gel. The progression of E.coli growth was significantly faster than on
plate 3 (LB mix1); this matched with other plate examples where the two strains were brought
into direct contact early. The E.coli cells rapidly expressed green florescence and spread across
the yeast lawn over a few days.
A liquid culture of both strains was incubated for several days then subjected to serial
dilution and plated (plate 10). An exact colony count was deemed inaccurate due to the lawn of
E.coli that had set up around the yeast. The yeast colonies were few in number but clearly visible
as a purple dot surrounded by white cells. The expression of green florescent protein by the
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E.coli was first witnessed on this plate in a colony growing between 3 colonies of S.cerevisiae
and pictured in image 1. From that point on the E.coli grew rapidly while expressing GFP in all
colonies adjacent to yeast colonies.

Image 1: spontaneous expression of green florescent protein by E.coli growing in proximity
to three S.cerevisiaecolonies. Photograph taken with an Olympus digital camera
mounted to a dissection scope.
Discussion:
Initial growth tests indicated a need to supplement the SAB medium with tryptone, which
is found in the LB medium that supports the growth of E.coli. On plate 3 (LBmix1) the yeast
were able to set up a lawn that did not get significantly penetrated by the E.coli throughout the
life of the plate despite media selection that should have favored E.coli growth. The E.coli
growing into the edge of the yeast lawn expressed GFP while the part of the lawn away from the
yeast failed to express it throughout the trial.
On plate 7 (SABmix1) the E.coli and S.cerevisiae came into direct contact with each
other. The E.coli rapidly spread across the top of the yeast lawn and rapidly expressed GFP. It is
interesting to note the E.coli grew faster across the yeast lawn than open media and expression of
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GFP occurred primarily in sections of the E.coli population in direct contact with yeast cells. The
yeast darkened over time turning from purple to brown.
In liquid cultures both strains were able to survive and produce colonies when transferred
to an agar plate. A possible explanation for this observation would be anoxic conditions at the
bottom of the test tube where both strains were being cultured. If an anoxic region were present it
would allow the S.cerevisiae to conduct anoxic fermentation on sugars found at that depth
creating a resource partition within the media.
The expression of green florescent protein in E.coli did not occur as expected. Plates
were produced using a prepared stock of L-arabinose yet the control plates did not induce
florescence. When the E.coli came in direct contact with the S.cerevisiae colonies, a rapid flush
of green could be seen spreading through the E.coli lawn. This could not be repeated on a plate
that contained no L-arabinose. The mechanism for this expression is unknown; it is possible the
yeast were taking up the sugar and passing it to the E.coli when brought into contact. Another
possibility would be a fermentation byproduct triggering GFP expression when it made its way
into the bacteria. Further studies into the flexibility of the pGLO plasmid or the minimum
concentration of L-arabinose required to induce GFP in this scenario would shed light on this
issue.


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Appendix A: Photographic series and media information
Plate 3: LB mix 1



Image
#
Date
Taken
Observations
1 5/3/12 Initial lawns setting up well, no GFP expression
2 5/4/12 No changes observed
3 5/7/12 White border along S.cerevisie= uracil exposure = dropped
plasmid
4 5/9/12 GFP expression along the white line = E.coli exposed to arabinose
5 5/10/12 Increased GFP expression along interface of both strains
6 5/14/12 GFP expression and robust growth spreading back from border
7 5/15/12 GFP expression now along formerly white areas = yeast attacked
8 5/17/12 Further E.coli growth, plate terminal

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Plate 7: SAB mix 1



Image
#
Date
Taken
Observations
1 5/3/12 Immediate contact observed, GFP expression at intersection
2 5/4/12 Rapid spread of E.coli along S.cerevisiaelawn; GFP expression
3 5/7/12 E.coli spread across the top of the yeast lawn
4 5/9/12 Purple color fading from yeast growing under E.coli
5 5/10/12 E.coli growth continues, yeast has not spread at all
6 5/14/12 E.coli colonies stable, yeast continues to lose ground
7 5/15/12 Darkening of the yeast colony, continued spread of E.coli
8 5/17/12 E.coli considered to have dominated plate, plate is terminal


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Plate 10: Serial Dilution of Hybrid culture


Image
#
Date
Taken
Observations
1 5/22/12 Colonies of E.coli and S.cerevisiaepresent from liquid culture; note
top left a white yeast colony.
2 5/24/12 No expression of GFP by E.coli, yeast colonies turning white
3 5/25/12 First visible evidence of GFP expression by E.coli
4 5/25/12 Closeup image of GFP expression by E.coli
5 5/29/12 GFP expression where E.coli colonies are adjacent to S.cerevisiae
6 5/31/12 E.coli seen growing into yeast colonies

Media recipes:
Media: LB Mix LB liquid mix SAB Mix SAB liquid mix
Sigma-Aldritch 5g LB .625g LB 6.00g SAB mix .75g SAB mix
4.00g Dextrose .5g Dextrose 2.00g Tryptone .25g Tryptone
3g Agar 3g Agar
200ml Water 25ml Water 200ml Water 25ml Water
Autoclave then: 400mg (2mg/ml)
L-arabinose
50mg
L-arabinose
400mg (2mg/ml)
L-arabinose
50mg
L-arabinose
Yield: 8 plates 25ml media 8 plates 25ml Media


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Literature Cited:
E. coli strain hb101 k-12. Bio-Rad Laboratories. Retrieved April 29, 2013, from
http://www.bio-rad.com/prd/en/US/LSR/SKU/166-0408EDU/E.-coli-Strain-HB101-K-12
pGLO
TM
Plasmid and GFP Kits. Bio-Rad Laboratories. Retrieved April 29, 2013 from
https://www.bio-rad.com/evportal/en/US/LSE/Category/f75948d2-dc20-4a32-b4e5-
b7e0fe4c21ed/pGLO-Plasmid-and-GFP-Kits
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broth .Journal of Bacteriology, 189(23), 87468749.
Keppler-Ross, S., Noffz, C., & Dean, N. (2008). A new purple fluorescent color marker for
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710.
Domingues, L., Lima, N., & Teixeira, J. (2000). Contamination of a high-cell-density continuous
bioreactor. Biotechnology and Bioengineering, 68(5), 584-587.
Verduyn, C., Postma, E., Scheffers , A., & Dijken, J. (1990). Physiology of saccharomyces
cerevisiae in anaerobic glucose-limited chemostat cultures. Journal of General
Microbiology, (136), 395-403.