Supplementary Information for Probiotics and Antimicrobial Proteins
-carotene biosynthesis in probiotic bacteria
Jennifer K. Miller1, M. Travis Harrison2, Annalisa D’Andrea2, Aaron N. Endsley1,
Fangfang Yin2, Krishna Kodukula1, Douglas S. Watson1,*
1
Biosciences Division, SRI International, Harrisonburg, Virginia 22802
2
Biosciences Division, SRI International, Menlo Park, California 94025
* To whom correspondence should be addressed. Tel: (540) 438-6600; Fax: (540) 438-6601; E-mail:
watson.douglas@gmail.com
Contents:
Supplementary Methods
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Methods
Generation of EcN-BETA Strain
Electrocompetent EcN were prepared as follows. An isolated EcN colony was
inoculated into 50 mL lysogeny broth (LB) and grown for 16 h at 37 oC with
shaking at 250 rpm (final OD600 4.6). 40 mL of this starter culture was inoculated
into 950 mL LB and grown at 37 oC with shaking at 250 rpm until OD600 0.6 was
reached. This culture was transferred to an ice water bath, cooled, pourted into ice
cold centrifuge bottles, and centrifuged at 2500 rpm for 30 min at 4 oC (RC-5C
Plus, Sorvall, Newtown, CT). Supernatants were decanted and pellets were
resuspended by pipetting in 500 mL ice cold, sterile double distilled H2O.
Centrifugation and resuspension was repeated in 250 mL double distilled H20,
followed by 10 mL sterile 10% glycerol. Contents of multiple centrifuge bottles
were then combined, centrifuged, and resuspended in 1 mL sterile GYT medium
(10% v/v glycerol, 0.125% w/v yeast extract, 0.25% w/v tryptone) with gentle
swirling. Aliquots of the cell suspension (50 L) were snap frozen in liquid N2
and stored at -80 oC.
EcN was transformed with pSTBlue-BETAipi as described below. Aliquots of
electrocompetent EcN, prepared as described above, were thawed on ice. Plasmid
(pSTBlue-BETAipi; 200 ng in 5 L) was added to electrocompetent cells and
stirred gently with a pipet tip to mix. The contents were transferred to an ice cold
electroporation cuvette (2 mm gap) and pulsed at 2 kV, 25 mF, 200 Ω (GenePulser X Cell Electroporator, Bio-Rad, Hercules, CA). Cells were diluted in 950
L SOC medium (Invitrogen) in a 1.5 mL microcentrifuge tube, incubated on ice
for 2 min, and further incubated for 3 h at 37 oC with shaking at 200 rpm. This
mixture (50 L) was streaked onto LB agar plates containing 100 g/mL
carbenicillin (‘selective LB agar plates’) and incubated at 37 oC overnight.
Individual transformed colonies were clearly identifiable by an orange color. EcN
was transformed with an empty vector plasmid lacking insert (pSTBlue1,
Novagen, Madison, WI) by an analogous method to generate a control strain
(EcN-VECTOR). To generate glycerol stocks, individual colonies were selected,
inoculated into 4 mL LB containing 100 g/mL carbenicillin (‘selective LB’), and
incubated overnight at 37 oC with shaking at 250 rpm. This overnight culture was
diluted with an equal volume of 50% (v/v) sterile glycerol, aliquotted, and frozen
at -80 oC.
The identity of the EcN strain and the presence of pSTBlue-BETAipi were
confirmed by PCR. Isolated colonies of EcN-BETA, untransformed EcN, E. coli
DH5, and E. coli K-12 were inoculated into 5 mL LB (or selective LB, for EcNBETA) and grown 16 h at 37 oC with shaking at 250 rpm. After 16 h, plasmid
DNA was isolated using a QIAprep spin Miniprep kit (Qiagen, Valencia, CA)
according to the manufacturer’s protocol. For PCR of isolated plasmid, DNA
template (50-90 ng) and primers (10 pM) were added to a PCR master mix in
thick walled 0.65 mL microcentrifuge tubes and PCR reactions were performed in
a PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA) using the
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following program: 94 oC for 2 min, repeat 29x (94 oC for 15 s, 50 oC for 30 s, 68
o
C for 30 s), 72 oC for 5 min. DNA polymerase was from Invitrogen (Platinum
Taq High Fidelity) and dTNPs were from Takara (Otsu, Shiga, Japan). Following
amplification, samples were run on a 1.0% agarose gel at 95 V (Power Pac 300,
Bio-Rad), stained with ethidium bromide, and imaged with a Chemi Doc™ XRS+
(Bio-Rad). Primers specific for EcN cryptic plasmid pMUT2 were identical to
those reported by Blum-Oehler et al. 1: F1 – 5’GACCAAGCGATAACCGGATG-3’; R1 – 3’-GTGAGATGATGGCCACGATT5’; F2 - 5’-GCGAGGTAACCTCGAACATG-3’; R2 - 3’CGGCGTATCGATAATTCACG-5’. Primers specific for pSTBlue-BETAipi
were designed from the insert sequence obtained during this work: F3 - 5’CCTGAGCGATCTAAAGCAGGCTGATA-3’; R3 - 3’GGGCTGACTTCAGGTGCTACATTT-5’. Expected sizes for these PCR
fragments were 427, 313, and 427 bp, respectively.
Spectrophotometric Assay of -carotene
-carotene was detected spectrophotometrically by its characteristic absorption
maximum near 460 nm 2. At the conclusion of bacterial growth experiments, cells
were harvested by centrifugation for 15 min at 3000 rpm (RT7, Sorvall) and
supernatants were removed to clean conical tubes. Tetrahydrofuran (THF; 3 to 5
mL) was added to each cell pellet and pellets were homogenized by vortexing
continuously for 2 min. Insoluble material was then pelleted by centrifugation for
15 min at 3000 rpm and supernatants were removed to clean conical tubes. In
control experiments, this procedure resulted in extraction of >95% of measurable
-carotene from bacterial cell pellets. The absorbance of each sample at 460 nm
was then recorded using a DU 640 spectrophotometer (Beckman Instruments,
Fullerton, CA). When necessary, bacterial extracts were further diluted in THF to
maintain absorbance values in the range of 0.1 to 1.1 absorbance units.
Absorbance at 600 nm was also recorded to check for turbidity and the presence
of residual insoluble material. As a negative control, -carotene was measured in
extracts of untransformed EcN by an analogous method. -carotene concentration
was calculated from the appropriate standard curves, obtained by preparing 2-fold
dilutions of pure -carotene in THF or in control bacterial extracts. Absorption
spectra of -carotene in THF, as well as THF extracts of both EcN and EcNBETA, were obtained using a SpectraMax Plus 384 (Carlsbad, CA). In some
experiments, the presence of -carotene in the culture supernatant was measured
using selective LB media as a blank. In these studies, absorbance values at 460
nm and 600 nm were measured before and after filtration of the culture media
through 0.45 m GHP Acrodisc GF syringe filters (Pall, Port Washington, NY).
EcN-BETA cultures were grown in the dark and care was taken throughout the
course of these experiments to minimize exposure to light.
The presence of -carotene in EcN-BETA was further confirmed by an LCMS/MS method by PharmaOn (Princeton, NJ). In brief, THF extracts of EcNBETA were prepared as described, dried by rotary evaporation, and further dried
under high vacuum overnight. Dried samples were solubilized in a mixture of
methyl tert-butyl ether and ethanol (1:4) and fractionated with a Shimadzu HPLC
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system (Columbia, MD) on an analytical C18 column (MAC-MOD HydroBondTM,
Chadds Ford, PA) using a water:acetonitrile:methanol gradient containing 1%
formic acid (v/v). HPLC eluant was analyzed by tandem mass spectrometry using
an AB 4000 Q TRAP (AB Sciex, Foster City, CA) with electrospray ionization in
positive mode. For these experiments, THF extracts of EcN-BETA containing at
least 75 g -carotene were combined and dried prior to solubilization and LCMS/MS analysis.
Stimulation of Murine Dendritic Cells with EcN-BETA
Murine dendritic cells were derived from the bone marrow of Balb/c mice
(Jackson Laboratories, Bar Harbor, ME). Femurs were harvested, the ends of the
bones were removed, and the femurs were flushed with PBS. The resulting cell
suspension was passed through a 70 µm strainer (BD Biosciences, San Jose, CA)
and erythrocytes were removed by hypotonic lysis in Tris-ammonium chloride
buffer (20 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, pH 7.5) for 4 min at room
temperature. Bone marrow cells were plated in flat-bottom 96 well plates in
RPMI-1640 medium (Invitrogen) supplemented with 10% FCS, 2 mM Glutamax,
and 50 U/mL penicillin and 50 µg/mL streptomycin sulfate.
To induce dendritic cell differentiation, cells were incubated for 5 d with 50
ng/mL IL-4 and 40 ng/mL GM-CSF (PeproTech, Rocky Hill, NJ) at 5% CO2 and
37°C in a humidified incubator. After this period, half of the medium was
removed and replaced with medium containing bacterial cell extracts or control
compounds at the following concentrations: LPS, 1 g/mL; RA, 1 M; carotene, 1 M; EcN-BETA, 0.4 M -carotene; EcN, 0.4 M -carotene
equivalent. BDMCs were then incubated for an additional 2 days and
supernatants and cells were collected for cytokine and flow cytometery analysis,
respectively. THF extracts were chosen for DC stimulation for several reasons.
First, THF extracts are amenable to spectrophotometric determination of βcarotene content, whereas bacterial lysates are either turbid or contain detergents
that interfere with spectrophotometric β-carotene determination. This permitted
cells to be treated with a defined amount of β-carotene. Second, extraction with
THF reduces the chemical complexity of the bacterial extract through removal of
components that are insoluble in THF, such as proteins, nucleic acids,
carbohydrates, and some lipids. Cytokine concentrations in BMDC culture
supernatants were quantified by ELISA according to the protocols provided by the
manufacturers. IFN-, IL-1, IL-12, IL-6, TNF-, and KC were measured with a
mouse 7-plex Th1/Th2 kit (Meso Scale Discovery, Gaithersburg, MD) using a
SECTOR Imager 2400 (Meso Scale Discovery). TGF-, IL-22, and IL-23 were
measured with kits from R&D Systems (Minneapolis, MN) using a PowerWave
HT 340 plate reader (BioTek Instruments, Winooski, VT).
For flow cytometry studies, BMDCs were suspended in PBS with 1% BSA (w/v)
and 0.05% sodium azide (w/v) and incubated with mouse IgG (Sigma) for 5 min
at 2-8°C before adding 0.2-1 µg of each antibody and further incubating for 30
min at 2-8°C. Antibodies Alexa 488 anti-CD1d, PerCP-eFluor 710 anti-MHC
Class II (I-A/I-E), PE-Cy7 anti-CD11c, PE anti-CCR9, and APC anti-CD40,
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along with appropriate isotype controls, were from eBioscience (San Diego, CA).
V450 anti-CD86 antibody and a corresponding isotype control were from BD
Biosciences (Franklin Lakes, NJ). Cells were then washed 3 times with PBS with
1% BSA and 0.05% sodium azide and analyzed on an LSR II flow cytometer (BD
Biosciences). All animal experiments were conducted under protocols approved
by SRI’s Institutional Animal Care and Use Committee in compliance with
appropriate regulations and policies regarding the humane treatment of animals.
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