of 49
Supporting Information
Chemoenzymatic L
abeling of
P
roteins for
Imaging in Bacterial C
ells
Samuel H. Ho and David A. Tirrell*
Division of Chemistry and Chemical Engineering, California Institute of Technology
Pasadena, California 91125
, United States
tirrell@caltech.edu
Table of Contents
Materials and Methods
................................
................................
................................
................................
................................
...............
1
Plasmid construction in pQE80
-
L
................................
................................
................................
................................
..............................
2
Table S1. Primers used in construction of pQE80
-
L
-
based vectors
................................
................................
................................
.........
2
Plasmid construction in pBAD24
................................
................................
................................
................................
...............................
2
Table S2. Primers used in construction of pBAD24
-
based vectors
................................
................................
................................
..........
3
Table S3.
E. coli
strains constructed in this study
................................
................................
................................
................................
......
4
Protein sequences used in this study
................................
................................
................................
................................
.........................
5
Gel analyses
................................
................................
................................
................................
................................
..............................
6
Protein expression and labeling with
ω
-
azido fatty acids
................................
................................
................................
...........................
6
Cell viability with com
pounds
1
3
................................
................................
................................
................................
..............................
7
Preparation of fatty acid methyl esters (FAMEs) and mass analysis
................................
................................
................................
.........
7
Lipid extraction from cells grown with
1
or
3
and mass analysis
................................
................................
................................
................
7
Fluorescence labeling in cell lysates
................................
................................
................................
................................
..........................
8
Immunoblotting
................................
................................
................................
................................
................................
...........................
8
Protein expression, purification, and mass
spectrometry of Tar, CheA, FtsZ, and FtsA
................................
................................
............
9
Table S4. Calculated and observed masses for modification of Tar
................................
................................
................................
........
12
Table S5. Calculated and observed masses for modification of CheA
................................
................................
................................
....
12
Table S6. Calculated and observed masses for modification of FtsZ
................................
................................
................................
......
12
Table
S7. Calculated and observed masses for modification of FtsA
................................
................................
................................
......
12
Labeling bacterial proteins with
ω
-
azido fatty acids
in fixed cells
................................
................................
................................
............
13
Labeling bacterial proteins with
ω
-
azido fatty acids in live cells
................................
................................
................................
...............
13
Fluorescence microscopy
................................
................................
................................
................................
................................
........
13
Discussion on fatty acid
probes
1
and
3
................................
................................
................................
................................
..................
13
Discussion on fatty acid pool and intact lipids in
E. coli
................................
................................
................................
...........................
14
Synthetic procedures
................................
................................
................................
................................
................................
...............
14
Synthesis of fatty acid probes
1
and
3
; and synthesis of BCN
BODIPY conjugate
2
................................
................................
............
14
Supplementary Figures
................................
................................
................................
................................
................................
............
17
Figure S1. Schematic of vector construction
in pQE80
-
L and pHV738
-
NMT
-
MetAP plasmids
................................
...............................
17
Figure S2. Fluorescence emission from labeled proteins expressed from pQE80
-
L plasmids
................................
................................
18
Figure S3. Deconvoluted mass spectra of Tar
................................
................................
................................
................................
........
19
Figure S4. Deconvoluted mass spectra of CheA
................................
................................
................................
................................
.....
20
Figure S5. Deconvoluted
mass spectra of FtsZ
................................
................................
................................
................................
.......
21
Figure S6. Deconvoluted mass spectra of FtsA
................................
................................
................................
................................
.......
22
Figure S7. Screening for fatty acid and fluorophore concentration ranges
................................
................................
..............................
23
Figure S8. Addition of
1
,
2
, or
3
does not affect cell growth
................................
................................
................................
....................
24
Figure S9. GC traces of FAMEs extracted fro
m
E. coli
................................
................................
................................
............................
25
Figure S10. LC traces of intact lipids from
E. coli
................................
................................
................................
................................
....
26
Figure S11. Identification of fatty acids from phospholipids (negative mode)
................................
................................
..........................
27
Figure S12. Identification of fatty acids from phospholipids (positive mode)
................................
................................
...........................
28
Figure S13
. In
-
gel fluorescence detection of proteins expressed from pBAD24 plasmids and labeled with
1
................................
........
29
Figure S14
. Immunofluorescence labeling of bacterial proteins
................................
................................
................................
..............
30
Figure S15
. Immunofluorescence labeling of uninduced cells or uninduced cells treated with
1
................................
............................
3
1
Figure S16
. Relative fluorescence intensities for live cells labeled with
1
or
3
and
2
................................
................................
..............
32
Figure S17. Cells can be labeled with lower concentrations of arabinose
................................
................................
...............................
33
Figure S18
. Live
-
cell fluorescence imaging of non
-
labeled cells
................................
................................
................................
.............
33
Figure S19
. In
-
gel fluorescence detection of proteins expressed from pQE80
-
L plasmids and labeled with
3
................................
.......
34
Figure S20
. In
-
gel fluorescence detection of proteins expressed from pBAD24 plasmids and labeled with
3
................................
........
35
Figure S21
. Fluorescence emission of proteins expre
ssed from pQE80
-
L plasmids and labeled with
3
................................
................
36
Figure S22
. Deconvoluted mass spectra of Tar
................................
................................
................................
................................
.......
37
Figure S23
. Deconvoluted mass spectra of CheA
................................
................................
................................
................................
...
38
Figure S24
. Deconvoluted mass spectra of FtsZ
................................
................................
................................
................................
.....
39
Figure S25
. Deconvoluted mass spectra of FtsA
................................
................................
................................
................................
.....
40
Figure S26. The lipidome of cells treated with
3
does not
differ from that of untreated cells
................................
................................
...
41
Figure S27
. Representative live
-
cell images for chemotaxis and cell division proteins labeled with
3
................................
....................
42
References
................................
................................
................................
................................
................................
...............................
43
Copies of NMR spectra
................................
................................
................................
................................
................................
............
44
S
1
Material
s
and Methods
.
Unless otherwise stated, all
synthetic
reactions were performed using
oven
-
dried glassware and under an atmosphere of argon. Anhydrous solvents (
N,N
-
dimethylformamide and
dichloromethane
) were purchased from Sigma
-
Aldrich and kept under
argon
. All other chemicals and reagents for chemical reactions were purchased from commercial
vendors (Sigma
-
Aldrich, Oakwood Chemical, Life Technologies) and used without further
purification. Reactions were monitored with thin layer chromatography (EMD/Merck
silica gel
60 F254 pre
-
coated plates) and UV light for visualization,
with
an acidic mixture of
phosphomolybdic acid, cerium ammonium molybdate, or basic aqueous KMnO
4
as developing
agents. Flash chromatography purifications were carried out usi
ng EMD/Merc
k silica gel 60
(230
400 mesh
).
1
H and
13
C NMR spectra were
measured
on either a
Bruker Prodigy 400
(400
MHz and 101 MHz, respectively) or a Varian Inova 500 (500 MHz and 126
MHz, respectively),
as noted.
1
H and
13
C NMR chemical shifts are reported as
δ
in units of parts per million (ppm)
relative to
tetramethylsilane (TMS
,
δ
= 0
) and calibrated using the
residual solvent peak in
chloroform
(
δ
7.26, singlet
and
δ
77.16, respectively). Data for
1
H NMR are reported as follows:
chemical shift (
δ
ppm), multi
plicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad), coupling constant (Hz), and integration. High
-
resoluti
on mass
spectrometry (HRMS) was
performed with either a JEOL JMS
-
600H High Resolut
ion Mass
Spectrometer with fast
atom bombardment (FAB) at the California Institute of Technology Mass
Spectr
ometry
Facility
in the Division of Chemistry and Chemical Engineering
or with an Agilent
6200 Series TOF with an Agilent G1978A Multimode source in electrospray ionization (ESI),
atmospheric
pressure
chemical ionization (APCI), or mixed (MM) ionization mode at the
Caltech
Beckman Institute
Center for Catalysis and Chemical Synthesis
.
Low
-
resolution mass
spectrometry (LRMS) was perfor
med using an Agilent 1290 UHPLC
MS in positive or
negative
mode at the Center for Catalysis and Chemical Synthesis.
Lysogeny broth (LB)
contained
10 g casein hydrolysate, 5 g yeast extract, and 10 g NaCl per
liter. Super optimal broth (SOB) was composed of 20 g casein hydrolys
ate, 5 g yeast extract,
0.5
8 g N
aCl, 0.19
g
KCl, 10 mL 1 M MgCl
2
, and 10 mL
1M MgSO
4
per liter. Super optimal
broth with catabolite repression (SOC) was comprised of SOB with the addition of 0.1 mL 2 M
glucose to 1 L of SOB medium.
Hyper Broth (
AthenaES
TM
, USA) was purchased and prepared
according to the manufacturer’s instructions.
Ampicillin
sodium salt
(
BioPioneer, USA
) and
kanamycin
sulfate
(
BioPioneer, USA
) were used at working concentration
s
of 200
μ
g/mL and 35
μ
g/mL, respectively. Phosphate
-
buffered
saline (PBS) was purchased from
Thermo Fisher
Scientific (USA)
and maintained at pH 7.4
.
PBST was a solution comprising PBS and 0.1% v/v
Tween
-
20.
Q5 Hot Start High
-
Fidelity DNA Polymerase, rest
riction endonucleases, Instant
Sticky
-
End Master M
ix,
Antarct
ic phosphatase,
and Blunt Ligase Master M
ix were all purchased
from New England Biolabs (
USA) and used
according to the manufacturer’s instructions
.
DNA
constructs were sequenced
by
Retrogen (USA).
Cells were made chemically competent by using the Mix & Go
E. coli
Transformation Kit
(Zymo Research, USA) according to the manufa
cturer’s instructions.
Electrocompetent cells
were freshly prepared as follows: 8 mL of overnight cultures were washed extensively with 300
mM sucrose on ice. The culture was then conc
entrated to a final volume of 60
μ
L cells, after
which 20
μ
L cells were
mixed with 50 ng
plasmid DNA. Electroporation was carried out using
the Gene Pulse XCell (BioRad, USA) according to the recommended manufacturer’s protocol.
Cells were immediately resc
ued in 500
μ
L SOC at 37
°C
for 1 h,
and
plated on LB agar plates
carrying the appropriate antibiotic supplements.
Plates containing colonies were always used
within one week for further experiments.
S
2
The plasmid encoding
N
-
myristoyltransferase (NMT) and
methionyl
aminopeptidase
(pHV738
-
NMT
-
MetAP)
was a generous gift from the Kahn laboratory
(Emory University, USA).
The plasmid confers resistance against kanamycin
and contains the p15A origin of replication
.
1
Plasmid construction
in
pQE80
-
L.
The pQE80
-
L v
ector (Qiagen, USA) confers resistance
to
ampicillin and
carries
a
6xHis tag following the
T5
promoter sequence
,
lac
operon
and ribosomal
binding site (RB
S).
To remove the 6xHis tag and RBS, p
QE80
-
L was linearized with
Eco
RI
and
Hind
III. The gene
fragment
encoding the
N
-
terminal
nona
pe
ptide sequence from eukaryotic
c
alcineurin B (hCaNB) was 5’
ATG GGT AAC GAA GCG TCT TAC CCG CTG
3’
(encoding the peptide sequence MGNEASYP
L
for NMT recognition
)
.
The gene encoding the
chemoreceptor Tar was amplified
from g
enomic DH10B
E. coli
DNA
using primers such that
the final
PCR
product read
EcoRI
::RBS::hCaNB::BamHI::tar::SacI::6xHis::HindIII
. This
PCR
product was digested with EcoRI
/HindIII and ligated into pQE80
-
L to
yield
plasmid pQE80
-
hCaNB
-
Tar
-
6xHis. For the remai
ning bacterial proteins (CheA, FtsZ, and FtsA
)
amplification
was carried out
using
genomic DH10B
E. coli
DNA
as the template. P
rimers were designed such
that the
PCR
products read
BamHI::gene of interest::SacI
. The
PCR
products were digested with
BamHI/Sac
I and ligated into pQE80
-
hCaNB
-
Tar
-
6xHis, which was also digested with
BamHI/SacI to
yield
plasmids pQE80
-
hCaNB
-
CheA
-
6xHis, pQE80
-
hCaNB
-
FtsZ
-
6xHis, and
pQE80
-
hCaNB
-
FtsA
-
6xHis.
These plasmids were transformed
via electroporation
into BL21
cells that already
harbored plasmid pHV738
-
NMT
-
MetAP
, generating strains SHH001
004
(Table S3).
Colonies were selected against ampicillin and kanamycin.
The primers used in this
study are summarized
in Table S1.
Table S1. Primers used in construction of pQE80
-
L
-
based
vectors.
Plasmid name
Forward (5’ to 3’)
Reverse (3’ to 5’)
pQE80
-
hCaNB
-
Tar
-
6xHis
ATATGAATTCGAGGAGAAA
TTAACTATGGGTAACGAAGC
GTCTTACCCGCTGGGATCCA
TGATTAACCGTATCCGC
TATAAGCTTCTAGTGAT
GGTGATGGTGATGGAGC
TCAAATGTTTCCCAGTTT
GGATC
pQE80
-
hCaNB
-
CheA
-
6xHis
ATATATGGATCCGTGAGCAT
GGATATAAGCGATTTTTATC
AGACATTTTTTGATGAA
ATATATGAGCTCGGCGG
CGGTGTTCGCCAT
pQE80
-
hCaNB
-
FtsZ
-
6xHis
ATATATGGATCCATGTTTGA
ACCAATGGAACTT
ATATATGAGCTCATCAG
CTTGCTTACGCAG
pQE80
-
hCaNB
-
FtsA
-
6xHis
ATATATGGATCCATGATCAA
GGCGACGGAC
ATATATGAGCTCAAACT
CTTTTC
GCAGCCAACT
Plasmid construction
in
pBAD24.
The pBAD24 vector contains an arabinose
-
inducible
promoter and confers resistance against ampicilli
n. The gene encoding the methyl aspartate
chemoreceptor Tar was amplified from genomic DH10B
E. coli
DNA using primers such that
the final
PCR
product read
EcoR
I
::hCaNB::SpeI::tar::SacI::6xHis::HindIII
. Both
the PCR
product and
pBAD24 were digested with EcoRI
/HindIII and the
resulting DNA fragments were
ligated to make pBAD24
-
hCaNB
-
Tar
-
6xHis. For the rem
aining bacterial proteins (CheA, FtsZ,
and FtsA, also amplified from genomic DH10B
E. coli
DNA), primers were designed such that
the final
PCR
products read
SpeI::gene of interest::SacI
. The
PCR
products were digested with
SpeI/SacI and ligated into pBAD24
-
hCaNB
-
Tar
-
6xHis, which was also digested with SpeI/SacI
S
3
to make the plasmids pBAD24
-
hCaNB
-
CheA
-
6xHis, pBAD24
-
hCaNB
-
FtsZ
-
6xHis, and
pBAD24
-
hCaNB
-
FtsA
-
6xHis.
To encode
the
C
-
terminal myc epitope tag
and remove the
C
-
terminal His tag,
phosphorylated
primers
were used for amplification, with
the pBAD24
-
hCaNB
-
gene of interest
-
6xHis as the template
. (The forward primer
s
for these four constructs are the
same and contain the
sequence
encoding the
myc
epitope tag, whereas
each
reverse primer
corresponds to
a
uniqu
e bacterial protein.)
The template was then digested with D
pnI, after
which
blunt
-
end ligation was performed to make the plasmids pBAD24
-
hCaNB
-
Tar
-
cmyc,
pBAD24
-
hCaNB
-
CheA
-
cmyc, pBAD24
-
hCaNB
-
FtsZ
-
cmyc, and pBAD24
-
hCaNB
-
FtsA
-
cmyc.
These plasmids were transfo
rmed
via electroporation
into BL21 cells that already
harbored
plasmid pHV738
-
NMT
-
MetAP, generating strains SHH005
012 (Table S3)
. Colonies were
selected against
both
ampicillin and kanamycin.
The primers used to design these constructs are
summarized
in
Table S2.
Table S2. Primers used in construction of pBAD24
-
based vectors
.
Plasmid name
Forward (5’ to 3’)
Reverse (3’ to 5’)
pBAD24
-
hCaNB
-
Tar
-
6xHis
ATATATGAATTCACCATGG
GTAACGAAGCGTCTTACCC
GCTGACTAGTATGATTAAC
CGTATCCGC
TATAAGCTTCTAGTGAT
GGTGATGGTGATGGAGC
TCAAATGTTTCCCAGTTT
GGATC
pBAD24
-
hCaNB
-
CheA
-
6xHis
ATATATACTAGTGTGAGCA
TGGATATAAGCGATTTTTA
TCAGACATTTTTTGATGAA
ATATATGAGCTCGGCGG
CGGTGTTCGCCAT
pBAD24
-
hCaNB
-
FtsZ
-
6xHis
ATATATACTAGTATGTTTG
AACCAATGGAACTT
ATATATGAGCTCATCAG
CTTGCTTA
CGCAG
pBAD24
-
hCaNB
-
FtsA
-
6xHis
ATATATACTAGTATGATCA
AGGCGACGGAC
ATATATGAGCTCAAACT
CTTTTCGCAGCCAACT
pBAD24
-
hCaNB
-
Tar
-
cmyc
GAGCTCGAACAAAAACTTA
TTTCTGAAGAAGATCTGTA
GAAGCTTGGCTGTTTTGG
AAATGTTTCCCAGTTTG
GATC
pBAD24
-
hCaNB
-
CheA
-
cmyc
GAGCTCGAACAAAAACTTA
TTTCTGAAGAAGATCTGTA
GAAGCTTGGCTGTTTTGG
GGCGGCGGTGTTCGCCA
TA
pBAD24
-
hCaNB
-
FtsZ
-
cmyc
GAGCTCGAACAAAAACTTA
TTTCTGAAGAAGATCTGTA
GAAGCTTGGCTGTTTTGG
ATCAGCTTGCTTACGCA
GG
pBAD24
-
hCaNB
-
FtsA
-
cmyc
GAGCTCGAACAAAAACTTA
TTTCTGAAGAAGATCTGTA
GAAGCTTGGCTGTTTT
GG
AAACTCTTTTCGCAGCC
AAC
S
4
Table S3.
E. coli
strains
constructed
in this study.
Name
Strain
Genotype
SHH001
BL21
AmpR, KanR
pQE80
-
hCaNB
-
Tar
-
6xHis
pHV738
-
NMT
-
MetAP
SHH002
BL21
AmpR, KanR
pQE80
-
hCaNB
-
CheA
-
6xHis
pHV738
-
NMT
-
MetAP
SHH003
BL21
AmpR, KanR
pQE80
-
hCaNB
-
FtsZ
-
6xHis
pHV738
-
NMT
-
MetAP
SHH004
BL21
AmpR, KanR
pQE80
-
hCaNB
-
FtsA
-
6xHis
pHV738
-
NMT
-
MetAP
SHH005
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
Tar
-
6xHis
pHV738
-
NMT
-
MetAP
SHH006
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
CheA
-
6xHis
pHV738
-
NMT
-
MetAP
SHH007
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
FtsZ
-
6xHis
pHV738
-
NMT
-
MetAP
SHH008
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
FtsA
-
6xHis
pHV738
-
NMT
-
MetAP
SHH009
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
Tar
-
myc
pHV738
-
NMT
-
MetAP
SHH010
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
CheA
-
myc
pHV738
-
NMT
-
MetAP
SHH011
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
FtsZ
-
myc
pHV738
-
NMT
-
MetAP
SHH012
BL21
AmpR, KanR
pBAD24
-
hCaNB
-
FtsA
-
myc
pHV738
-
NMT
-
MetAP
S
5
Protein sequences used in this study.
The NMT recognition sequence
is
highlighted in
blue
.
C
-
terminal epitope tags
(
either 6xHis
or myc
)
are highlighted in
red
.
pQE80
-
hCaNB
-
Tar
-
6xHis
MGNEASYPL
GSMINRIRVVTLLVMVLGVFALLQLISGSLFFSSLHHSQKSFVVSNQLREQQGELTSTWDLMLQT
RINLSRSAVRMMMDSSNQQSNAKVELLDSARKTLAQAATHYKKFKSMAPLPEMVATSRNIDEKYKNYYTALTE
LIDYLDYGNTGAYFAQPTQGMQNAMGEAFAQYALSSEKLYRDIVTDNADDYRFAQWQLAVIALVVVLILLVAWY
GIRRMLLTPLAKIIAHIREIAGGNLANTLTIDGRSEMGDLAQSV
SHMQRSLTDTVTHVREGSDAIYAGTREIAAGN
TDLSSRTEQQASALEETAASMEQLTATVKQNADNARQASQLAQSASDTAQHGGKVVDGVVKTMHEIADSSKKI
ADIISVIDGIAFQTNILALNAAVEAARAGEQGRGFAVVAGEVRNLASRSAQAAKEIKALIEDSVSRVDTGSVLVES
AGETMNNIVNAVTRVTDIMGEIASASDEQSRGIDQVALAVSEMDRVTQQNASLVQESAAAAAALEEQASRLTQ
AV
SAFRLAASPLTNKPQTPSRPASEQPPAQPRLRIAEQDPNWETFEL
HHHHHH
pQE80
-
hCaNB
-
CheA
-
6xHis
MGNEASYPL
GSVSMDISDFYQTFFDEADELLADMEQHLLVLQPEAPDAEQLNAIFRAAHSIKGGAGTFGFSVL
QETTHLMENLLDEARRGEMQLNTDIINLFLETKDIMQEQLDAYKQSQEPDAASFDYICQALRQLALEAKGETPS
AVTRLSVVAKSEPQDEQSRSQSPRRIILSRLKA
GEVDLLEEELGHLTTLTDVVKGADSLSAILPGDIAEDDITAVL
CFVIEADQITFETVEVSPKISTPPVLKLAAEQAPTGRVEREKTTRSNESTSIRVAVEKVDQLINLVGELVITQSMLA
QRSSELDPVNHGDLITSMGQLQRNARDLQESVMSIRMMPMEYVFSRYPRLVRDLAGKLGKQVELTLVGSSTE
LDKSLIERIIDPLTHLVRNSLDHGIELPEKRLAAGKNSVGNLILSAEHQGGNICIEVTDDGAGL
NRERILAKAASQG
LTVSENMSDDEVAMLIFAPGFSTAEQVTDVSGRGVGMDVVKRNIQKMGGHVEIQSKQGTGTTIRILLPLTLAILD
GMSVRVADEVFILPLNAVMESLQPREADLHPLAGGERVLEVRGEYLPIVELWKVFNVAGAKTEATQGIVVILQS
GGRRYALLVDQLIGQHQVVVKNLESNYRKVPGISAATILGDGSVALIVDVSALQAINREQRMANTAAEL
HHHHH
H
pQE80
-
hCaNB
-
FtsZ
-
6xHis
MGNEASYPL
GSMFEPMELTNDAVIKVIGVGGGGGNAVEHMVRERIEGVEFFAVNTDAQALRKTAVGQTIQIGS
GITKGLGAGANPEVGRNAADEDRDALRAALEGADMVFIAAGMGGGTGTGAAPVVAEVAKDLGILTVAVVTKPF
NFEGKKRMAFAEQGITELSKHVDSLITIPNDKLLKVLGRGISLLDAFGAANDVLKGAVQGIAELITRPGLMNVDFA
DVRTVMSEMGY
AMMGSGVASGEDRAEEAAEMAISSPLLEDIDLSGARGVLVNITAGFDLRLDEFETVGNTIRAF
ASDNATVVIGTSLDPDMNDELRVTVVATGIGMDKRPEITLVTNKQVQQPVMDRYQQHGMAPLTQEQKPVAKV
VNDNAPQTAKEPDYLDIPAFLRKQADEL
HHHHHH
pQE80
-
hCaNB
-
FtsA
-
6xHis
MGNEASYPL
GSMIKATDRKLVVGLEIGTAKVAALVGEVLPDGMVNIIGVGSCPSRGMDKGGVNDLESVVKCVQ
RAIDQAELMADCQISSVYLALSGKHISCQNEIGMVPISEEEVTQEDVENVVHTAKSVRVRDEHRVLHVIPQEYAI
DYQEGIKNPVGLSGVRMQAKVHLITCHNDMAKNIVKAVERCGLKVDQLIFAGLASSYSVLTEDERELGVCVVDI
GGGTMDIAVYTGGALRHTKVIPYAGNVVTSDIAYAFGTPPSDA
EAIKVRHGCALGSIVGKDESVEVPSVGGRPP
RSLQRQTLAEVIEPRYTELLNLVNEEILQLQEKLRQQGVKHHLAAGIVLTGGAAQIEGLAACAQRVFHTQVRIGA
PLNITGLTDYAQEPYYSTAVGLLHYGKESHLNGEAEVEKRVTASVGSWIKRLNSWLRKEFEL
HHHHHH
pBAD24
-
hCaNB
-
Tar
-
cmyc
MGNEASYPL
TSMINRIRVVTLLVMVLGVFALLQLISGSLFFSSLHHSQKSFVVSNQLR
EQQGELTSTWDLMLQT
RINLSRSAVRMMMDSSNQQSNAKVELLDSARKTLAQAATHYKKFKSMAPLPEMVATSRNIDEKYKNYYTALTE
LIDYLDYGNTGAYFAQPTQGMQNAMGEAFAQYALSSEKLYRDIVTDNADDYRFAQWQLAVIALVVVLILLVAWY
GIRRMLLTPLAKIIAHIREIAGGNLANTLTIDGRSEMGDLAQSVSHMQRSLTDTVTHVREGSDAIYAGTREIAAGN
TDLSSRTEQQASALEET
AASMEQLTATVKQNADNARQASQLAQSASDTAQHGGKVVDGVVKTMHEIADSSKKI
ADIISVIDGIAFQTNILALNAAVEAARAGEQGRGFAVVAGEVRNLASRSAQAAKEIKALIEDSVSRVDTGSVLVES
AGETMNNIVNAVTRVTDIMGEIASASDEQSRGIDQVALAVSEMDRVTQQNASLVQESAAAAAALEEQASRLTQ
AVSAFRLAASPLTNKPQTPSRPASEQPPAQPRLRIAEQDPNWETFEL
EQKL
ISEEDL
pBAD24
-
hCaNB
-
CheA
-
cmyc
MGNEASYPL
GSVSMDISDFYQTFFDEADELLADMEQHLLVLQPEAPDAEQLNAIFRAAHSIKGGAGTFGFSVL
QETTHLMENLLDEARRGEMQLNTDIINLFLETKDIMQEQLDAYKQSQEPDAASFDYICQALRQLALEAKGETPS
AVTRLSVVAKSEPQDEQSRSQSPRRIILSRLKAGEVDLLEEELGHLTTLTDVVKGADSLSAILPGDIAEDDITAVL
S
6
CF
VIEADQITFETVEVSPKISTPPVLKLAAEQAPTGRVEREKTTRSNESTSIRVAVEKVDQLINLVGELVITQSMLA
QRSSELDPVNHGDLITSMGQLQRNARDLQESVMSIRMMPMEYVFSRYPRLVRDLAGKLGKQVELTLVGSSTE
LDKSLIERIIDPLTHLVRNSLDHGIELPEKRLAAGKNSVGNLILSAEHQGGNICIEVTDDGAGLNRERILAKAASQG
LTVSENMSDDEVAMLIFAPGFSTAEQVTDVSG
RGVGMDVVKRNIQKMGGHVEIQSKQGTGTTIRILLPLTLAILD
GMSVRVADEVFILPLNAVMESLQPREADLHPLAGGERVLEVRGEYLPIVELWKVFNVAGAKTEATQGIVVILQS
GGRRYALLVDQLIGQHQVVVKNLESNYRKVPGISAATILGDGSVALIVDVSALQAINREQRMANTAAEL
EQKLIS
EEDL
pBAD24
-
hCaNB
-
FtsZ
-
cmyc
MGNEASYPL
GSMFEPMELTNDAVIKVIGVGGGGGN
AVEHMVRERIEGVEFFAVNTDAQALRKTAVGQTIQIGS
GITKGLGAGANPEVGRNAADEDRDALRAALEGADMVFIAAGMGGGTGTGAAPVVAEVAKDLGILTVAVVTKPF
NFEGKKRMAFAEQGITELSKHVDSLITIPNDKLLKVLGRGISLLDAFGAANDVLKGAVQGIAELITRPGLMNVDFA
DVRTVMSEMGYAMMGSGVASGEDRAEEAAEMAISSPLLEDIDLSGARGVLVNITAGFDLRLDEFETVGN
TIRAF
ASDNATVVIGTSLDPDMNDELRVTVVATGIGMDKRPEITLVTNKQVQQPVMDRYQQHGMAPLTQEQKPVAKV
VNDNAPQTAKEPDYLDIPAFLRKQADEL
EQKLISEEDL
pBAD24
-
hCaNB
-
FtsA
-
cmyc
MGNEASYPL
GSMIKATDRKLVVGLEIGTAKVAALVGEVLPDGMVNIIGVGSCPSRGMDKGGVNDLESVVKCVQ
RAIDQAELMADCQISSVYLALSGKHISCQNEIGMVPISEEEVTQEDVENVVHTAKSVRVRDEHRVLHVIPQEYAI
DYQEGIKNPVGLSGVRMQAKVHLITCHNDMAKNIVKAVERCGLKVDQLIFAGLASSYSVLTEDERELGVCVVDI
GGGTMDIAVYTGGALRHTKVIPYAGNVVTSDIAYAFGTPPSDA
EAIKVRHGCALGSIVGKDESVEVPSVGGRPP
RSLQRQTLAEVIEPRYTELLNLVNEEILQLQEKLRQQGVKHHLAAGIVLTGGAAQIEGLAACAQRVFHTQVRIGA
PLNITGLTDYAQEPYYSTAVGLLHYGKESHLNGEAEVEKRVTASVGSWIKRLNSWLRKEFEL
EQKLISEEDL
Gel analyses.
Sodium dodecyl sulfate
poly(acryl
amide) gel electrophoresis (S
DS
PAGE) was
accomplished on a FB3000Q protein gel electrophoresis apparatus from Fisher Scientific (
USA
)
using NuPAGE Novex 4
12% Bis
-
Tris protein gels (1.0 mm, 10
-
well) from Thermo Fisher
(USA). Protein samples were mixed with an SDS loading buffer consi
sting of 0.05%
w
/v
bromophenol blue, 0.1 M dithiothreitol (DTT), 10%
w
/v glycerol, 2%
w
/v sodium dodecyl
sulfate, and 8 M urea in 50 mM Tris
,
pH 8.0. Samples were heated at 95
°
C for 10 min
to ensure
denaturation and reduction of disulfide bonds. SeeBlue P
lus 2 (Thermo Fisher, USA) was used
as a mole
cular weight marker for all SDS
PAGE experiments. Total protein loading was
visualized by using InstantBlue (Expedeon, Carlsbad, USA) as a
coomassie
blue stain, following
the manufacturer’s instructions.
Protein expression and labeling
with
ω
-
azido
fatty acids.
Individual colonies were
used to
inoculate
LB medium supplemented with 200
μ
g/mL ampicillin and 35
μ
g/mL kanamycin
,
and
cultures were
grown
overnight
at
37
°C with mild agitation (250 rpm
).
Cultures
w
ere diluted
1:
50 in 10 mL LB medium (also
supplemented with 200
μ
g/mL ampicillin and 35
μ
g/mL
kanamycin
)
and
grown until the optical density at 600 nm (OD
600
) reached 0.5
.
Protein
expression was induced with e
ither 1 mM
isopropyl
-
β
-
D
-
1
-
thiogalactopyrannoside (
IPTG
)
or
0.2%
w
/v
L
-
(+)
-
arabinose
(for pQE80
-
L or pBAD24 vectors, respectively)
. At the time of
induction, 500
μ
M of either
1
or
3
was added to the culture
; growth was continued
for an
additional
4 h
(
37
°C,
250 rpm
)
. Cultures
were harvested
, washed twice with PBS,
and
resuspended in a solution
containing
1% w/v sodium dodecyl sulfate (SDS) in Tris pH 8.4. Lysis
was accomplished by boiling cells at
90
°
C for 5
min, after which the solution was
cooled on ice
.
Benzonase
nuclease (Sigma
-
Aldrich, USA) and protease inhibitor (cOmplete, EDTA
-
free, Roche,
USA) were added to the lysate to
degrade
chromosomal DNA and to prevent protein proteolysis
,
respectively
. The lysate was allowed to incubate at
37
°C
for 1 h, after which t
he lysates were
centrifuged at 13,000 rpm at
4
°C
for 30 min. The supernatant was collected, and protein
S
7
quantification was performed using the bicinchoninic acid (BCA) assay kit (Thermo Fisher,
USA). Lysates were separated into aliquots and stored at
-
80
°C
until further use.
Protein
expression and labeling with fatty acids were performed in three independent replicate
experiments.
Cell viability with compounds 1
3.
Individual colonies were used to inoculate LB medium
supplemented with 200
μ
g/mL ampicilli
n and 35
μ
g/mL kanamycin, and cultures were grown
overnight at
37
°C with mild agitation (250 rpm). Cultures were diluted to an OD
600
of 0.1 in LB
medium (also supplemented with 200
μ
g/mL ampicillin and 35
μ
g/mL kanamycin). To each
culture was added 250
μ
M
1
or
3
, or 20
μ
M fluorophore
2
. Cultures were allowed to continue to
grow in the presence of each compound, and OD
600
was monitored every 10 min for 800 min
using a Varioskan LUX microplate reader (Thermo Fisher, USA). The OD
600
of cultures grown
without the presence of
1
3
were
also
measured as a control.
Preparation of fatty acid methyl esters (FAMEs) and mass analysis
.
Individual colonies
were used to inoculate LB medium supplemented with 200
μ
g/mL ampicillin and 35
μ
g/mL
kan
amycin, and cultures were grown overnight at
37
°C with mild agitation (250 rpm). Cultures
were diluted 1:50 in 10 mL LB medium (also supplemented with 200
μ
g/mL ampicillin and 35
μ
g/mL kanamycin) and grown until OD
600
reached 0.5.
To each culture was adde
d 250
μ
M
1
or
3
, or no fatty acid mimic as a control. Growth was continued (
37
°C, 250 rpm) until OD
600
reach
ed
approximatel
y 1.0. Cultures were harvested, washed twice with 0.5X PBS, and once
with ddH
2
O before being lyophilized. Fatty acids were extracted
and methylated in a single
acidic methylation step.
2
Briefly, freeze
-
dried cell pellets (approximately 8.0 mg) were
resuspended in a 20: 1 v/v methanol: acetyl chloride solution and heated at 100
°C
for 10 min.
The solutions were allowed to cool to room t
emperature, after which 1 mL hexane and 1 mL
water were added, and FAMEs were extracted twice with hexane (2 x 1 mL). The organic layers
were combined and concentrated to a final volume of 200
μ
L for mass analysis. GC
MS analysis
of FAMEs was accomplished
using a ThermoFinnigan Trace GC equipped with a HP
-
5MS
column (30 m x 0.250 mm x 0.25
μ
m), with the column effluent split between a flame ionization
detector (FID) and ThermoFinnigan DSQ mass spectrometer. The GC oven was held at 90
°C
for
1 min, ramped at
3
°C
/ min to 140
°C
, ramped at 3
°C
/ min to 250
°C
, and finally ramped at
20
°C
/ min to a final temperature of 310
°C
.
Known FAMEs present in
E.
coli
were identified
using the NIST MS Search 2.0 program and quantified against an internal standard (m
eth
yl
behenate
). GC traces of FAME extracts of cells not treated with fatty acids were compared with
GC traces of FAME extracts of cells treated with fatty acids to identify new peaks.
Lipid extraction from cells grown with 1 or 3 and mass analysis
.
Individu
al colonies were
used to inoculate LB medium supplemented with 200
μ
g/mL ampicillin and 35
μ
g/mL
kanamycin, and cultures were grown overnight at
37
°C with mild agitation (250 rpm). Cultures
were diluted 1:50 in 10 mL LB medium (also supplemented with 200
μ
g/mL ampicillin and 35
μ
g/mL kanamycin) and grown until OD
600
reached 0.5. To each culture was added 250
μ
M
1
or
3
, or no fatty acid mimic as a control. Growth was continued (
37
°C, 250 rpm) until OD
600
reach
ed
approximatel
y 1.0. Cultures were harvested
and washed twice with 0.5X PBS. Lipid
extraction was performed following literature protocols.
3
Briefly, cell pellets were resuspended
in 120
μ
L 0.1% wt/v ammonium acetate (dissolved in ddH
2
O). To the solution was added 300
μ
L methanol and 1 mL methyl
-
tert
-
butyl ether
(MTBE)
. Solutions were briefly vortexed and
S
8
then
sonicated for 1 hour at room temperature using a sonicator bath.
8
μ
g of internal standard
(di17:0 phosphatidylcholine, phosphatidyl
ethanolamine, and phosphatidyl
glycerol
)
(Avanti Polar
Lipids,
Alabaster, USA) was added at
this time.
Internal standards were prepared as 0.1 g/L
solutions in methanol/dichloromethane (9:1)
Lipids were extracted by addition of 260
μ
L of
water, forming a biphasic solution, with lipids in the top (MTBE/methanol) layer.
The
MTBE/methanol layer was separated from the water layer and dried under a gentle stream of
nitrogen.
Dried samples were dissolved in 1 mL 9:1 methanol/dichloromethane for analysis
using LC
MS. Fatty acids
1
and
3
were also dissolved to a final concentr
ation of 10
μ
M in 9:1
methanol/dichloromethane as standards for mass spectrometry.
Samples were submitted to the
Caltech Environmental Analysis Center
of the Beckman Institute
.
Mass spectrometry (UPLC
MS) analysis was
accomplished using an Acquity
I
-
Class UPLC coupled to a Xevo G2
-
S TOF
mass spectrometer (Waters, USA).
Lipid samples (injection volume, 5
μ
L)
were separated on an
Acquity UPLC CSH C18 column (2.1
μ
m
x 100 mm, 1.7
μ
m, Waters, USA). UPLC
TOF
MS
E
data
were collected in positive and negat
ive mode using electrospray ionization
(ESI).
Indiscriminate fragmentation
of
all
parent ions
was performed with the capillary voltage set to
2.0 KV, the cone voltage set to 30 V, and the acquisition range set for data collection between
100 to 2000 m/z.
M
S
E
data consists of two channels acquired simultaneously.
Extracted ion
chromatograms were performed using m/z searches based on fragment ions with a mass window
of 10 mDa.
Lipids were identified from their fragmentation patterns in both negative and posit
ive
mode.
Fluorescence labeling in cell lysates.
To 100
μ
g protein in cell lysate diluted to 250
μ
L with
PBS,
was added iodoacetamide to a
final concentration of 1
mM. Lysates were incubate
d
at
37
°C
for 1 h in the dark. Then,
2
was added
to each
solution to a final concentration of
2
μ
M.
Reac
tions were performed
at room temperature for
10 min
in the dark. Proteins were then
precipitated
by
addition of chloroform and methanol. Pellets were resuspended in SDS loading
buffer.
Samples were boiled for
10 min
at 95 °
C, after which 20
μ
g lysate were
subjected to
SDS
PAGE. Protein gels were washed with a solution containing 10% acetic acid, 20%
methanol, and 70% water, with mild agitation (2 x 30 min).
Fluorescence
labeling
was visualized
using a Typhoon T
rio (GE Healthcare, USA) with excitation at 488 nm, the photomultiplier tube
(PMT) voltage set to 400 V, and emission
monitored using a 520 nm band pass
filter
.
Protein
bands corresponding to the fluorescently labeled protein of interest were quantified us
ing
ImageQuant TL (GE Healthcare, USA), and normalized against the intensity of
that particular
band in
the coomassie lane.
Fluorescence labeling was quantified for each biological replicate
.
Plots representing the average fluorescence
intensities
from independent experiments were
prepared
using IGOR Pro (Wavemetrics, Oregon, USA).
Immunoblotting.
Protein
lysate
s
were
separated
by
SDS
PAGE and transferred to a 0.2
μ
m
nitrocellulose membrane using an iBlot 2 gel transfer apparatus from Life Technolo
gies (USA),
following the manufacturer’s protocol. Membranes were blocked with 5% w/v no
n
-
fat dry milk
in PBST at room temperature for 1 h
, and washed three times with PBST b
efore incubating with
either 1:
5000 PentaHis
Alexa
Fluor 647 (Qiagen, USA), or 1:
2
000 c
myc Alexa Fluor 647
conjugate (Ce
ll Signaling Technologies, USA) at
4
°
C
overnight
. Membranes
(covered from
light)
were then wash
ed five times with PBST
at room temperature
(10 min
each wash).
Fluorescence was visualized using a Typhoon Trio (GE Healthcare, USA) with excitation at 633
nm, the photomultiplier tube (PMT) voltage set to 300 V, and emission
monitored using
a 670
nm band pass
filter
.