Characterizing a Purple Pigment Defect
and Its Microbial Origin in Cheese Rinds
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Noelani Kamelamela
A Thesis in the Field of Biotechnology
for the Degree of Master of Liberal Arts in Extension Studies
Harvard University
May 2018
Abstract
Surface- ripened cheeses have a clear pattern of microbial succession which
contributes to the overall quality of the product. These rinds typically contain the visible
products of microbial activity including pigments, which contribute heavily to the
aesthetics of the rind. Most of these pigments have not been matched to their microbial
producer or producers. By reconstructing cheese rind microbial communities in the lab, I
recreated an unexpected purple pigment originally observed in a production cheese farm.
This purple rind defect is an interesting microbial pigment problem that has not yet been
characterized. Two microbes of the same phylum create similar pigments, a mixture of
indigo and indirubin, when cultured solo or together. The pigments were isolated and
identified by thin layer chromatography. Other phylogenetically diverse microbial
species were also able to produce purple pigments, suggesting this could be a common
trait in cheese rind microbiomes. These investigations provide new insights into pigment
iv Frontispiece
Author’s Biographical Sketch
Noelani Kamelamela currently works at their alma mater MIT at the BioMicro
Center as a Technical Associate who specializes in next generation sequencing projects.
They began their Harvard Extension School degree while working at Harvard University
in the Bauer Center and continued their studies while working at Tufts University as a
Lab Manager in Dr Benjamin Wolfe’s Lab. They have a diverse skillset which spans
vi Dedication
To my dear friends and family members for their unwavering support: lots of
hours of Skype, in person interactions, your gifts, texts and phone calls have kept me
Acknowledgments
This wouldn’t be possible without the direction of my thesis director and mentor
in this project: Dr Benjamin Wolfe. When I worked at Harvard University, he was a
post-doctoral fellow in the Dutton Lab who saw potential in me. I became his technician
with little microbiology experience when he became a Professor at Tufts University and
he helped me transition to my current position at the MIT BioMicro Center. He was
naturally the first person I thought of when I began the thesis process. His support of this
work, including bouncing ideas off of each other at all hours over email, meetings in
person and the occasional pun or two have kept me interested in honing my ability to ask
specific questions in microbial systems. Many members of the Wolfe lab past and present
have contributed ideas, encouragement, and advice, most notably during this project:
Kaite Zhang, Brittany Niccum, Esther Miller, and Liz Landis. I am also grateful to my
supervisors, colleagues, coworkers and coops at the BioMicro Center, most notably
during this project: Stuart Levine, Jon Penterman, Austin Hendricks and Meghan
viii
Table of Contents
Frontispiece ... iv
Author’s Biographical Sketch ...v
Dedication ... vi
Acknowledgments... vii
List of Tables ...x
List of Figures ... xi
I. Introduction ...1
Pigments in Cheese Rinds: Orange to Purple ...5
Purple Rind Defect ...8
II. Materials and Methods ...10
Standard Media and Culturing Techniques...10
Extraction of Pigments from Spent Media...12
Thin Layer Chromatography...13
Indole Spot Assay ...13
16S rRNA Sanger sequencing for Identification and Phylogenetic Comparison ..14
III. Results ...15
IV. Discussion ...25
Purple Pigment is a Combination of Metabolites ...26
Microbial Warfare: Purple Pigment is a Poison ...28
Study Limitations ...31
Future Research Directions ...32
Conclusion ...34
Appendix Supplemental Table and Figure ...35
x List of Tables
Table 1. Standard media for culturing and experiments………...11
List of Figures
Figure 1. Potential origins of cheese microbes (Wolfe et al., 2014) ...3
Figure 2. Washed rind cheese wheels displaying variety of purple rind defect ...8
Figure 3. Dilutions of raw-milk cheese on BHI ...16
Figure 4. In vitro identification of pigments created by P .vulgaris, P .faecalis or both on
PCAMS with indoxyl sulfate and tryptophan ...19
Figure 5. Separation by centrifugation...21
Figure 6. In vitro identification identification of pigments created by both species
together on CCA with indoxyl sulfate and tryptophan. ...22
Figure 7. Different pigmentation in community co-culturing experiments on CCA. ...23
Figure 8. Indigoid production with either Tryptophan, Indole or Indoxyl Sulfate as a
precursor ...27
Figure 9. Phylogenetic tree of 16S rRNA sequences with purple pigment producers on
Chapter I.
Introduction
For thousands of years, people have fermented raw materials to preserve foods
producing pickled meats, vegetables and other foodstuffs. Also created through
fermentation are drinks such as wine, sake, beer, kombucha and coffee. Fermented foods
are also sought after as tasty additions to meals. Cabbage can ferment into kimchi, a spicy
Korean side dish, or sauerkraut, a sour tasting German condiment. One of the most
widely appreciated products of fermentation is cheese (Wolfe & Dutton, 2015).
Cheesemaking has a rich history, and the methods and end products vary around
the world. Traditional methods utilize the coagulation of the milk protein casein to create
solid curds (Fox et al., 2017). The solid curds can be gathered from the liquid mass
known as whey. Pressed together, these curds form a solid body of cheese. A cheese rind
arises during the aging process, an outside layer which may contain post-fermentation
microbial activity. Fresh cheeses and soft cheeses tend not to have discernible rinds and
are typically very pliable and moist. These cheeses are “fresh” because they are not
usually aged beyond either pressing curd together or working the curd free of whey
since soft cheeses are sold moist and should be consumed once purchased (W.
Bockelman, 2010).
Starter cultures can be added to the initial coagulation products, but cheese
commensal diversity on the surface can be altered through addition of microbes, control
of pH, moisture, temperature and salinity as the cheese ages (Button and Dutton, 2012).
Diverse fungal and bacterial communities can contribute to the final properties of the
product, like smell, taste, and color (Irlinger et al., 2015). Variation in production
methods encourage the growth of different communities of microbes or inhibit the growth
of specific undesirable species (Donnelly et al., 2014).
Microbial species found in cheese rinds can be derived from a variety of sources
as shown in Figure 1. If coming from unpasteurized dairy, even the cow or cowhand can
be a potential source. Starter cultures, typically purchased commercially to ensure even
and expected succession of cheese microbes can be added as the curds are formed. Salt
may harbor microbes as well, sometimes from their processing plant or, if directly
created from seawater, possibly from the ocean. The facility in which a cheese ages may
3
Figure 1. Potential origins of cheese microbes (Wolfe et al., 2014)
Cheese curds, post curdling and separation from whey, tend to have a pH of
around 5. Microbes, which can thrive in this lower pH environment, such as those of the
genus Staphylococcus, tend to colonize the surface of the cheese first. Following
de-acidification by these first microbes, the pH of the surface then rises towards 7 allowing
for the appearance of Actinobacteria such as Brevibacterium and Brachybacterium. In the
middle stages of succession, molds such as Penicillium and or Geotrichum may
successfully thrive. Collectively, these organisms form a thick biofilm over time, which
is commonly called the rind (W. Bockelman, 2010).
There are three major categorizations of naturally aged raw milk cheeses which
are surface-ripened cheeses: washed rind, natural rind and bloomy rind. Bloomy rinds
and a dense rind. Natural rinds are primarily uninoculated, which means that the main
drivers of microbial diversity stem primarily from the starter culture as well as the aging
environment. As a washed rind cheese is aged, the rind is washed in salt water
periodically, which promotes the growth of communities that normally survive at certain
salinities on the rind, which are primarily bacteria accustomed to both salt and moisture
(Wolfe et al., 2014). The aging processes of a cheese plays a large role in succession of
microbes on its rind.
A washed rind cheese may be initially cultured in the same methods as both a
natural or bloomy rind cheese (Wolfe et al., 2014). They are treated with regular salt
solution washes during the rest of the aging process may drastically alter the microbiome.
Each wash may add specific microbes to the initial microbiome, select for populations,
which can tolerate changes in salinity or pH or moisture.
Typical microbial members of a washed rind cheese, also known as smear ripened
cheese, include yeasts such as Debaryomyces hansenii and Geotrichum candidum. These
types of yeasts as well as known early bacterial colonizers of cheese rinds such as the
Firmicute Staphylococcus may de-acidify a cheese rind. Proteobacteria which are
halotolerant, such as Vibrio, Halomonas, Psychrobacter and Pseudoalteromonas are
typically widespread in these cheeses since as marine associated bacteria they may be
5
Psychrobacter and Proteus in these cheeses is not unexpected, but may not be desirable
since both can create some very pungent smells (Detae et al., 2008). Proteus vulgaris
may not be typically identified in most cheeses, but has been sampled from cheese aged
in high moisture and high salinity (Fox et al., 2004).
Pigments in Cheese Rinds: Orange to Purple
The appearance of the cheese is highly dependent on the rind microbiome as milk
components such as proteins like casein, sugars like lactose, fattty acids, immunoglobins,
amino acids, various vitamins, and essential minerals are used or turned into byproducts.
Only raw milk will contain active enzymes such as lipases, lactoperoxidase, and lactase
as well as biologically active proteins such as lactoferrin (Huppertz T et al., 2009).
Ordinary pigmentation of raw milk cheese rinds can vary, since the rind is a biofilm that
can only successfully form upon succession of many colonies of microbes. Any
noticeable changes in cheese texture, smell, rind coloration, can be indicative of changes
in succession or unexpected dynamic interactions between rind members.
Bloomy and natural rind cheeses tend to have rinds which are less moist than
washed rind cheeses due to the presence of molds. Typical rind coloration of washed
rind cheeses range from a yellowish orange to a yellowish white with light pink tones
(Bockelmann, 2010). If a washed rind cheese is washed in a solution with color, such as
a red wine or brown beer, additional pigments may remain on the surface. Actinobacteria
distinct orange of a washed rind cheese (Galaup et al., 2015). Pigments in cell walls will
tend to remain within colonies of similar microbes while secreted pigments may migrate
on the surface of the rind away from the microbes which produced them.
Typical molds found on naturally aged cheeses include Geotrichum candidum and
Penicillium camemberti, both of which provide white and grey coloring to the surface of
a rind. Penicillium candidum presents as fully white, and is typically dominant in
bloomy rind cheeses. Penicillium roqueforti is inoculated to the curds pre-aging and can
also be added to the body of the cheese during aging to allow the spores within to spread
and grow in blue cheeses (Michelson, 2010).
Previous studies have explored the microbial origins of pigment defects in cheese.
For example,the discovery of blue pigments produced by bacteria in cheese rinds have
been studied as safety issues and curiosities (Martin et al., 2011; Andreani et al., 2015;
Caputo et al., 2015). In the case of Mozzarella blue discoloration, the pigment resided
within the liquid ,which the cheese is stored in. Eventually, the blue pigment permeated
the entire piece of Mozzarella. In order to identify the pigment or pigments creating the
blue color, thin layer chromatography with a control was typically done and identified the
blue pigment as indigo. Culturing techniques and simple methods of identification
through microscope or 16S rRNA sequencing pinpointed for each study a single blue
7
been shown to produce blue pigment, but other strains also do not when grown in the
same conditions.
Chromobacterium violescens is the first described and most studied bacterial
species known to have purple colored cell walls (Lichstein and Van De Sand, 1945) and
C. violescens is not typically found in cheese. Purple pigment discoloration of the cheese
appeared similar in color to images purple urine bag syndrome (Dealler et al., 1988) as
well as in description of the production of indigo and indirubin, which mixed together
look purple, in wastewater (Zhang et al., 2015). Two possible compounds, which can
create a similar color in other microbiomes are indigo and indirubin together. In studies
of purple urine bag syndrome, Providencia stuartii and Klebsiella pneumoniae were
assumed to be potential solo producers of purple pigments (Dealler et al., 1988).
The origins of many cheese rind pigments are unknown. Each cheese rind has a
variety of microbial members present. An individual bacterial species can make a
specific pigment or set of pigments. Another possibility is that two or more species could
make unique pigments through interactions with each other. Primarily, as explained
above, individual pigments are most easily linked to the cell wall of a single bacterial
species. Interactions between microbial members of the rind community may confound
Purple Rind Defect
The overall goal of this thesis is to characterize and better understand the
microbial origins of a previously uncharacterized cheese rind pigment defect. The Wolfe
Lab has been studying a particular washed rind cheese. This cheese exhibited an
unplanned purple discoloration also known as a purple rind defect depicted in Figure 2.
The cheesemaker had not changed any particular aspect of production prior to the
discovery of purple appearing on a cheese rind. Eventually, more than one cheese rind in
each batch had a purple hue. We assisted this cheesemaker by identifying the member or
members of the rind responsible for the shift in color.
Figure 2. Washed rind cheese wheels displaying variety of purple rind defect
Samples of raw milk, the cheese with the purple rind defect pre-aging, and cheese
with the purple rind defect were given to the lab. All bacterial communities sampled
9
raw milk. Individuals from the community were cultured from the preserved glycerol
stocks. Single colonies were cloned, and the 16S ribosomal RNA (16S rRNA) genes
were amplified, Sanger sequenced, and compared to the NCBI database for identification
purposes.
All rind samples contained both Proteus and Psychrobacter. Other members of
the ordinary rind community, which were culturable and identifiable by 16S sequencing
include Staphylococcus equorum, Vibrio literalis and Psychrobacter cibarius. The
dominant culturable species are Proteobacteria or the Staphylococcus. Rind areas with
the purple pigment had Proteus vulgaris and Psychrobacter faecalis while ordinary
non-purple regions did not have both species present. Neither of these bacteria are typically
purple, as both strains from the purple rind areas produced colonies that range from
creamy white to mauve on standard growth medium. Once we had identified the
microbes responsible, we proposed the work of this thesis. Our goals were to identify the
pigment in vitro and to identify whether the ability of microbes to create pigments in
Chapter II.
Materials and Methods
Molecular biology techniques and experimental protocols used throughout the
study are outlined within this section. All experiments were conducted in sterile
conditions under either a flame or in a biosafety cabinet. Although the majority of our
concerns lie within biology, compound characterization relied also on analytical
chemistry and visual observation. Briefly, our microbes of interest were cultured in
catabolite repression media. When provided precursors in the indigo and indirubin
biosynthesis pathway, two microbes produced purple pigments. Pigments were identified
by thin layer chromatography. Using the same type of media, other
Gammaproteobacteria yielded mixed results with and without pigment production.
Phylogenies were constructed for comparison of the results of the screen.
Standard Media and Culturing Techniques
Tryptophan at 4.8 mM and indoxyl sulfate at 7.9 mM, which were used to induce
indigo production for Providencia stuartii and Klebsiella pneumoniae in liquid culture
(Dealler et al., 1988), were added to autoclaved standard media after sterile filtration of
stock solutions. Both solid and liquid media were used. No antibiotics were used to
11
Table 1. Standard media for culturing and experiments
Media Ingredients (per 1 L of liquid) Brain Heart Infusion (BHI)
pH 7
7.7 g Calf Brain (infusion from 200 g) 9.8 g Beef Heart (infusion from 250 g) 10.0 g Proteose Peptone
2.0 g Dextrose 5.0 Sodium Chloride 2.5 g Disodium Phosphate [15 g Agar for solid media] Plate Count Agar with Milk and Salt (PCAMS)
pH 5
5.0 g Tryptone 2.5 g Yeast Extract 1.0 g Dextrose
1.0 g Whole Milk Powder 10.0 g Sodium Chloride [15.0 g Agar for solid media] Cheese Curd Agar (CCA)
pH 5
100.0 g Freeze-dried Cheese Curd 5.0 g Xanthan Gum
30.0 g Sodium Chloride [17.0 g Agar for solid media]
Initial isolation of microbes from the cheese rind were accomplished by scraping
the surface of the rind with a sterile dowel and placing a small amount into 1xPBS.
Several dilutions of the scraping were plated onto BHI plates. Colonies were then used to
create larger solo glycerol stocks in 15% Glycerol diluted with 1xPBS. Lab culture
techniques were employed to regrow communities from glycerol stocks which were
stored properly at -80 ℃. Glycerol stocks were thawed at 4 ℃ for at least 30 minutes prior to dilution and inoculation in or on standard growth media. Sterile glass beads
were shaken across the surface of the plate after cells were added. To estimate
onto standard BHI plates and colonies were counted within a week of plating. Typical
concentrations for our glycerol stocks were around 106 colony forming units (CFUs) per
1 microliter. Communities of microbes were plated on CCA solid media to test whether
specific microbes or communities caused purple rind defect.
Extraction of Pigments from Spent Media
Harvesting of plates to extract pigment involved moving the agar of the plate with or
without cells into a WhirlPak™ bag, adding sterile deionized water equivalent to one
third the weight of the plate, and homogenizing using a 50 mL conical tube on a flat
bench surface. Liquids or solids were moved from the bag into 5 mL centrifuge tubes
and centrifuged at maximum speed for 10 minutes. Supernatant was filter sterilized into
1.5 mL microcentrifuge tubes with a 0.2 uM syringe filter using 1 mL syringes.
Sterilized supernatant was frozen at -20 C or -80 C before being placed in the Thermo
Savant SC110A Speedvac Concentrator for about 8 hours on High to concentrate the
extractions. This freeze was also considered sterilizing since there was no glycerol to
buffer the cells from lysis. Occasionally, sterile filtration and dehydration was skipped
when the amount of pigmented liquid extracted with these methods was very low.
Methanol was used for extraction with vigorous vortexing for about 1 min followed by a
light centrifugation. Extract was used for TLC as well as given to a collaborator for
13
Thin Layer Chromatography
Thin layer chromatography (TLC) is a relatively inexpensive method of
separating and identifying compounds and has been used to identify indigoid compounds
in microbial systems before (Rui et al., 2005; Tyihak et al., 2016). To identify indigoid
pigments TLC with chloroform 97% and acetone 3% as the mobile phase was performed
as described in a similar indigoid production system utilizing Escherichia coli (G. Cairo,
2015) with silica as the stationary phase. The silica plate was a Uniplate™ 46521 (silica
gel HL with organic binder) from Analtech. Plates were activated by placing in a drying
oven at 110 °C for at least 1 hour. Methanol was added to the extractions instead of
chloroform for safety reasons. After vigorous vortexing and centrifugation, the methanol
added extraction could be added to the plate and the TLC plate could be developed in a
covered Coplin jar with a strip of filter paper (WhatMan Qualitative Cellulose 1005-125)
to saturate the atmosphere in the jar. The positive controls were indigo and indirubin: a
cospot of both controls were used since co-elution did not present signs of interference.
TLC has not been previously reported on indigoid compounds with either species of
interest.
Indole Spot Assay
Glycerol stocks of each microbe of interest were streaked on BHI using a sterile
dowel. After the growth of colonies stabilized for about a week, plates were placed in a
4°C fridge. A cotton swab was applied to gather various cells. Two drops of Spot Indole
monitored for color change over 30 mins. Swabs which change from an ordinary colony
color to purple or blue were determined to have microbes which rapidly produce indole.
16S rRNA Sanger sequencing for Identification and Phylogenetic Comparison
Glycerol stocks of each microbe of interest were streaked on BHI using a sterile
dowel. After the growth of colonies stabilized for about a week, plates were placed in a
4°C fridge. DNA was extracted with the Qiagen DNeasy® PowerSoil® Kit (12888-100)
with 50 uL for the final elution instead of 100 uL. A dilution of 1:10 of the final elution
is taken for amplification in a standard Polymerase Chain Reaction with bacterial 16S
primers 27f and 1492r (J.A. Frank et al., 2003). PCR products were confirmed by
standard gel electrophoresis in 1% agarose with 1xTAE buffer at 120V/200mA for 30
minutes. Use of SYBR Safe DNA stain allowed for UV visualization of the products
with a 100 bp ladder with the Bio-RAD UV Imager. Products were sent to GeneWiz
with primer 27f for Sanger sequencing . Sequences were trimmed and submitted to
Megablast in Geneious to confirm identity at the species level. An alignment of a 450 bp
region of the 16S rRNA gene was made using MUSCLE and a phylogenetic tree was
Chapter III.
Results
Both P. faecalis and P. vulgaris strains from the cheese with the purple rind
defect were isolated with four different strains to obtain pure cultures. Two BHI plates of
different dilutions are shown in Figure 3. Four different strains taken from the non purple
rind sections were identified by 16S rRNA sequencing as two strains of Brevibacterium
linens, Staphylococcus equorum and Marinilactibacillus sp. Marinilactibacillus spp. are
lactic acid bacteria ,which are usually found in the curd pre-aging. S. equorum is a
Firmicute and early colonizer. Both M. sp and S. equorum colonies appear white and
nearly transparent when grown on standard growth media. Brevibacterium linens appears
as the pH of the rind begins to de-acidify and these two strains were orange and yellow
when grown on standard growth media. These microbes might be representative of
different times of succession with P. faecalis and P. vulgaris strains appearing last on the
Figure 3. Dilutions of raw-milk cheese on BHI
All species are found in the left plate, the section cultured from non-purple cheese, while only P. faecalis and P.vulgaris cultured from purple cheese form colonies in the plate on the right.
Indoxyl sulfate and tryptophan are precursors for pathways which produce
indigoids indigo and indirubin that can lead to purple pigment on the cheese. Indoxyl
sulfate is not necessarily produced by either member, but can be found in milk
(Klostermeyer et al., 1985). Similarly, tryptophan may not be synthesized by either
member, but can be found in milk in high enough amounts to induce tryptophanase
activity (USDA, 2017). In order to induce tryptophanase activity, we introduced both a
higher amount of tryptophan and indoxyl sulfate than average into culturing media to
17
Liquid cultures of both P. vulgaris and P. faecalis from the purple rind defect
cheese in Brain Heart Infusion broth (BHI), Plate Count Agar with Milk and Salt broth
(PCAMS) and Cheese Curd broth (CCA) with and without both indoxyl sulfate and
tryptophan did not produce noticeable amounts of indigoids when incubated
conventionally on a shaker at room temperature, in darkness at room temperature and in a
cheese-cave like environment, which was covered from light at a temperature of 15
℃. Primary growth at room temperature and then movement to a cheese cave like environment also was not productive. Visibly there was no red or purple color appearing
over time although a biofilm of cells did form on the the surface of liquid
cultures. Dilutions of these cultures and liquid media were also inspected for changes in
color.
In the cheese-cave like environment, PCAMS plates with tryptophan and indoxyl
sulfate did become visibly pink and then brown over time after the formation of a
biofilm, a visible collection of either microbial cell bodies or byproducts, with either P.
faecalis, P. vulgaris and both species together when compared to the control plates as
well as other tested species taken from the ordinary rind. These observations indicated
that formation of a biofilm were a precursor to color change in the first week post
inoculation. Primary growth at room temperature and then movement to the cheese cave
Simple homogenization in sterile deionized water produced pigmented liquid
which could quickly be dehydrated and then extracted with methanol or simply extracted
with methanol for TLC. In Figure 4, the most visibly colorful results of several runs are
presented. Pigments extracted from plates with both P. vulgaris and P. faecalis included
both indigo and indirubin. Spots corresponding to extractions are separated into at least
three compounds from PCAMS and at least three compounds from CCA; a few of the
spots must be indirubin and indigo because the blue spot is at the same position as the
indigo standard spot and the pink-purple spot corresponds with the indirubin standard
19
Figure 4. In vitro identification of pigments created by P .vulgaris, P .faecalis or both on
PCAMS with indoxyl sulfate and tryptophan
a. Comparison of color between control plate, P .vulgaris, P .faecalis or both seven days post inoculation. b. TLC run optimized for the camera. The cospot of Indigo and Indirubin is furthest left while extracts from PCAMS with indoxyl sulfate and tryptophan with P. vulgaris, P. faecalis, are labelled Pr, Ps and PrPs respectively.
Reproduction of the pigments on PCAMS+IS+T media was successful. I then
moved on to test if the pigments could be produced on a more cheese-like medium,
CCA. Curds are separated from whey and immediately frozen, dehydrated and
homogenized into small flakes. Cheese Curd Agar plates with Tryptophan at 4.8 mM and
Indoxyl sulfate at 7.9 mM also became visibly pink over time after the formation of a
biofilm with either P. vulgaris or both species together after approximately a month in
the cheese cave. Both P. vulgaris and P. faecalis did not grow as prolifically alone on the
CCA plates, which are typically pH 5. Primary growth at room temperature and then
movement to the cheese cave was productive. Overall growth at room temperature did
not result in pigmentation.
Homogenization in sterile deionized water produced a mixture, which could not
be dehydrated and then extracted with methanol successfully. Separation was then
undertaken with rounds of centrifugation on homogenized mixtures instead. Figure 5
illustrates the end product of the CCA+IS+T extraction with P. vulgaris and P. faecalis,
and both together. Ethanol was added directly to the mixtures or to the supernatant of the
21 Figure 5. Separation by centrifugation
Centrifugation at maximum for 20 minutes or longer would create a pigmented liquid layer for plates, which had visible pigmentation
In Figure 6, the most visible result of several runs are presented. Pigments
extracted from plates with both P. vulgaris and P. faecalis possibly included both indigo
and indirubin. The image on the left in 6a is standard exposure and the image on the
right is blacklight or UV-A exposure. There is a visible indirubin spot travelling from
the P. vulgaris and P. faecalis spotted extract. Under standard lighting conditions, no
visible indigo spot can be seen for either spotted extracts. Under blacklight exposure,
there appears to be a spot migrating to the same distance as indigo, but only for the P.
vulgaris and P. faecalis spotted extract. Quantitatively, Rf values could not be calculated
Figure 6. In vitro identification identification of pigments created by both species
together on CCA with indoxyl sulfate and tryptophan.
a. Comparison of color between control plate, P .vulgaris, P .faecalis or both one month post inoculation. b. TLC run optimized for camera under regular light and
blacklight. The cospot of Indigo and Indirubin is furthest left while extracts from CCA with indoxyl sulfate and tryptophan with P. vulgaris and P. faecalis together and P. vulgaris alone, are labelled PrPs and Pr respectively. There were several development runs, this run was optimized for the camera.
23
non-purple members of the purple rind community was attempted. Various pigments
appeared, as shown in Figure 7. Purple coloration did appear within the community
cultured CCA in some cases, but not for all cases. Purple pigmentation was not observed
in the control treatment of just the 4 non-purple members. Only one of each treatment
with P.faecalis or P. vulgaris appeared to have purple pigmentation. Extraction attempts
were deemed failures since extracts did not migrate at all during TLC. One potential
cause was that other byproducts were much higher in concentration than indigo or
indirubin, which were also produced in the CCA.
Figure 7. Different pigmentation in community co-culturing experiments on CCA.
From left to right: control community, control community with P. faecalis, control community with P. vulgaris and control with both P. faecalis and P. vulgaris.
Recreation of the purple pigment on both types of cheese-like media lead us to
question whether this behavior was common with all Proteobacteria from cheese or
common to the other community members of the purple pigmented rind. A selection of
thirteen Proteobacteria in the laboratory culture collection and the four other community
members were taken and cultured on PCAMS+IS+T in a cheese cave like
environment. Purple pigment was observed in 5 strains from the genera Proteus and
Psychrobacter including P. vulgaris and P. faecalis from the purple rind. Results of this
screen are detailed in Table 2 which appears in the Appendix.
Images were collected as evidence for the screen at different timepoints over a
month and the screen was performed at separate times to confirm the appearance of
purple pigment over a week in a cheese cave like environment. Few of the microbes
from the lab culture collection were purple and none of the non-purple microbes from the
purple defect rind created purple pigment. A phylogenetic tree was constructed to
compare the results of this screen with the groupings of microbes in Figure 8 (also in the
Appendix). It was not confirmed with TLC that microbes which created purple pigment
were actually making indigo and indirubin although it is likely. Independent validation
with liquid chromatography-mass spectroscopy also validated the presence of indigo and
Chapter IV.
Discussion
The aim of this study was to identify the purple rind defect presented to the Wolfe
Lab and the microbial member or members creating the purple rind defect. In order to
fulfill these goals, the purple rind defect was recreated in vitro and extracted by various
methods for analysis by thin layer chromatography. An additional aim was to identify if
other microbes of the same phylum or the common microbes within the cheese of interest
could create the purple pigment under similar conditions.
The literature cited indicated that the mechanism by which purple pigment was
created was likely metabolism of tryptophan into indigoids. The evidence collected
strongly suggests that the presence of tryptophan and or indoxyl sulfate were necessary
for indigoid production by either P. vulgaris or P. faecalis. The production of indigoids
only with additional tryptophan and indoxyl sulfate in media strongly support the
proposed pathway.
None of the four other rind community microbes produced purple pigment under
the same conditions. Of the Proteobacteria selected and tested in similar ways, only four
a phylogeny, it was apparent that purple pigment production on cheese like media in a
cheese cave environment was limited to certain Proteobacteria.
Purple Pigment is a Combination of Metabolites
The pathways in which indigo and indirubin are both byproducts tend to involve
the synthesis of indole by tryptophanase and then the metabolization of indole into
indigoid compounds. This metabolic pathway becomes necessary when other sources of
carbon and energy are unavailable, also known as catabolite repression. Figure 9 below,
adapted from (Dealler et al., 1988), illustrates a pathway through which tryptophan can
be utilized as a source of both carbon and energy, and end products include indigoid
compounds with the underlying assumption is that microbes will have transport
mechanisms for the substrates and the products. Enzymes which should be encoded into
the genes are shown in the figure and are listed as: tryptophanase, sulfotransferase,
phosphatase and sulphatase. A single organism does not need to have all three or four
27
Figure 8. Indigoid production with either Tryptophan, Indole or Indoxyl Sulfate as a
precursor
(adapted from Dealler et al., 1988)
Tryptophan is a precursor, thus a tryptophanase analog or related genes should be
identifiable in the bacterial genome of a potential producer of indole. Indole is a signal
peptide which can be produced and utilized by most bacteria (Lee et al., 2010). Not all
bacteria can produce indole, but even bacteria which do not produce indole may be
2005), thus a potential producer of indigo and indirubin will likely have a sequenceable
oxygenase analog or related genes. Tryptophan in the case of cheese must be locked up
in the casein, but we were unable to investigate the mechanism by which tryptophan was
transported into the cells. It is possible that some microbes could make tryptophan
available for degradation with proteases while not metabolizing tryptophan or that some
microbes can do both.
A genomic and transcriptomic approach has been successful in a different cheese
system to investigate a similar issue (Andreani et al., 2015). In addition to doing a
genomic study, they completed a transcriptomic study with RNA Sequencing which
measures gene expression by sequencing messenger RNA. Although a transcriptomic
approach is beyond the scope of this thesis, I think it is important to note that genomic
implementation in this project may lay the groundwork for future investigations.
Microbial Warfare: Purple Pigment is a Poison
Pigment production by microbes can span the entire color wheel. It is implied
that such pigments are created by these organisms for a variety of purposes: metabolism
in the case of chlorophyll production, protection from antioxidation with carotenoids, and
many more. Another potential purpose for synthesizing non-metabolized molecules
compound is to increase the stability of an organism or group versus other organisms or
29
Different communities of microbes may produce indigoid compounds in different
amounts. The production of indigo is not necessarily related to whether a community can
produce a these compounds or not. Although cheese rinds are biofilms, these films are
typically of the same pigments created by the dominant organisms, whether fungi or
bacteria or a mixture of both. In our studies, the color exists throughout the biofilm, but
does not penetrate deeply into the body of the cheese. The compound could easily be
diluted into water or methanol which is typical of indigo.
Related to human waste, the production in human urine bags by bacteria has been
a fascinating subject of study. Historically, the presence of a blue color in urine bags was
a cause for alarm, but it was not necessarily an indication of a disease for the human
attached to it. The trajectory from indole precursors in human urine to indigoids was
traced and ultimately determined to have a bacterial cause, although determination was
made through looking at cell morphologies (Dealler et al., 1988). A more recent case
study within a nursing home found that it was common in a small sample population, but
not harmful since urine bags did not provide flow of urine back to the patient (Lin et al.,
2010). Biofilms have not been identified in any urine bags related to purple urine bag
syndrome.
In the microbial world, indigo production may be protective of the producer since
it has the potential to rid the immediate ecosystem of antagonists. On the other hand,
indole is necessary for certain bacterial species as a signal peptide since it can determine
2010). Oxygenases may also act upon indole to create indigoids. Non-indole producing
bacteria, plants and animals can produce these oxygenases. The production of indigo in
this case makes competition harder for bacteria that require indole to function.
Genomic Investigation into Indigoid Production Pathways
As mentioned previously, oxygenases metabolize indole during the synthesis of
indigoids. Tryptophanases can also take precursors along known pathways in
microbiomes to facilitate indole synthesis. I consulted various open source online
resources such EnsemblBacteria, the National Center for Biotechnology Information
(NCBI) and European Nucleotide Database to identify genomes similar to the isolates of
interest from the purple cheese. Proteus vulgaris sequences available online at NCBI
appear to have a few tryptophanase and tryptophanase analogs, but not necessarily
oxygenase analogs. On the other hand, Psychrobacter faecalis has no available full
genome as well as only several partial sequences and few annotations available in NCBI.
A tryptophanase operon of wild type P. vulgaris has been cloned in Escherichia
coli and Sanger sequenced (Kamath & Yanofsky, 1992). By culturing P. vulgaris in
catabolite repression medium, researchers selected for colonies of P. vulgaris with high
trytophanase activity relative to the wild type. Our isolate may prove to have these
operons and yet still not produce purple pigment which would be similar to the results of
31
Psychrobacter colonies and strains produce purple pigment and others do not. All Proteus strains we tested did produce purple pigment in catabolite repression medium.
Our phylogenetic comparison among similar species were correlated with
experimental data to hypothesize whether there exist common genes for tryptophanases
or oxygenases within certain groupings of microbes within a cheese rind. Our cheese
rind of interest does also have other microbes and as a community members could be
acting in concert, independently or at cross purposes. We are capable of using this
comparison to see that certain Proteobacteria are more likely to have these tryptophanases
or oxygenases than others.
Study Limitations
TLC results and LC-MS results from a collaborator verified that P. vulgaris, P.
faecalis, or P. vulgaris and P. faecalis can produce the purple pigment on cheese like
media in a cheese cave like environment. When both species were present, pigmentation
appeared a day or two before appearance in solo culture. Pigment was more concentrated
in co-culture than in solo culture. Two producers with the same precursors and same
active pathway could be making twice as much pigment. It was not clear whether there
were negative or positive interactions between P. vulgaris and P. faecalis. It was also not
clear as to whether the presence of the pigment had a negative or positive effect on any of
In community culture and solo culture, in the case of P. faecalis, pigmentation
was not always present. The four other community strains are canonically better at early
colonization and may have exhausted available resources before these two Proteobacteria
could successfully thrive. P. faecalis may require the assistance of a related microbe
such as P. vulgaris to thrive in a cheese like environment. Also, the formation of a
biofilm or at least a high enough amount of colonies covering the surface of the cheese
like media also was required for production of the purple pigment in solo cultures. The
motility of P. vulgaris and other Proteus strains is higher than non-Proteus strains in this
study. P. vulgaris colonies spread rapidly when it is the only microbe on the surface of
cheese like media. In the presence of the other PMC microbes, it was hard to visibly
identify P. vulgaris colonies.
Future Research Directions
Expanding on these results would involve delving into the relationship between P.
faecalis and P. vulgaris on cheese. Since P. vulgaris is more motile than P. faecalis,
perhaps P. vulgaris may initially colonize and spread forming a biofilm which could
allow P. faecalis to attach and thrive on the surface of the cheese. Our results do not
suggest more than this commensalism between these two strains. With both possibly
possessing the same active tryptophan metabolism pathway there also may be nutrient
33
Another related topic is the direct effect of indigo and indirubin on community
succession. A 16S population sequencing study identified that different microbial
communities of wastewater may produce different amounts of indigo and also a variety
of structures from different precursors. Shifts in community composition were detected
during indigo production, but predictions in community shift could not be made on the
basis of production timing or amount alone (Zhang et al., 2015). The production of
indigoids in this case appear to be shifting the population on the purple rind towards
indigoid producers, disrupting expected community stability.
To disrupt the formation of a purple rind defect, a cheesemaker should consider
reducing the salt content of their brine or changing the salt source entirely if they spot a
purple rind amongst their cheeses. Indigo is considered to be toxic and hazardous for
human consumption (NCBI, 2017), so the use of the Proteobacteria of interest in cheese
is only useful if cheese is being made for scientific study not for human
consumption. An additional control would be to keep cheese more dry than normal by
reducing humidity where the cheese is being aged. A potential action to reduce the
amount of proteobacteria in the Psychrobacter or Proteus Genera from succeeding on the
rind would be to intentionally add high proportions of halotolerant bacteria known to be
common and safe on cheese rinds such as Brevibacterium linens or Staphylococcus
I did not have the opportunity to determine the genes that control pigment
production, especially in Psychrobacter where indigo production has not previously been
reported. I have been able to suggest mechanisms and potential genes of interest. Thus,
this thesis may lay a foundation for those future investigations.
Conclusion
We hypothesized that P. faecalis and P. vulgaris were the key microbial strains
making purple pigment on a raw milk washed rind cheese and that the purple pigment
was a byproduct of tryptophan metabolism. Purple pigment was recreated with both
strains present on cheese like media in a cheese cave like environment in vitro. The
pigment was identified by TLC to be byproducts of tryptophan metabolism, indigo and
indirubin. Both purple pigment producing strains were proven to be more closely related
Appendix
Supplemental Table and Figure
Table 2. Screen for Purple Pigment on PCAMS+IS+T
Genus species Tree Purple
Proteus vulgaris Pr X Psychrobacter faecalis Ps X Brevibacterium linens 1 Staphylococcus equorum 2 Marinilactibacillus 4 Brevibacterium linens 7 Hafnia JB232 Halomonas neptunia H8 Halomonas JB37 Proteus vulgaris JB425 X Psychrobacter BP6263 X Psychrobacter H1 Psychrobacter glacincola H9 Psychrobacter JB193 Psychrobacter maritimus RH1 X Psychrobacter glacincola RH4 Serratia proteamaculans W11 Serratia proteamaculans BW106 Vibrio JB196
Figure 9. Phylogenetic tree of 16S rRNA sequences with purple pigment producers on
CCA in purple
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