Postsynthetic modification of bacterial peptidoglycan using bioorthogonal n-acetylcysteamine analogs and peptidoglycan o-acetyltransferase B

Article abstract of DOI:10.1021/jacs.7b06820

Bacteria have the natural ability to install protective postsynthetic modifications onto its bacterial peptidoglycan (PG), the coat woven into bacterial cell wall. Peptidoglycan O-acetyltransferase B (PatB) catalyzes the O-acetylation of PG in Gram (-) bacteria, which AIDS in bacterial survival, as it prevents autolysins such as lysozyme from cleaving the PG. We explored the mechanistic details of PatB's acetylation function and determined that PatB has substrate specificity for bioorthgonal short N-acetyl cysteamine (SNAc) donors. A variety of functionality including azides and alkynes were installed on tri-N-acetylglucosamine (NAG)3, a PG mimic, as well as PG isolated from various Gram (+) and Gram (-) bacterial species. The bioorthogonal modifications protect the isolated PG against lysozyme degradation in vitro. We further demonstrate that this postsynthetic modification of PG can be extended to use click chemistry to fluorescently label the mature PG in whole bacterial cells of Bacillus subtilis. Modifying PG postsynthetically can aid in the development of antibiotics and immune modulators by expanding the understanding of how PG is processed by lytic enzymes.

Full text of DOI:10.1021/jacs.7b06820

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Communication
Post-synthetic modification of bacterial peptidoglycan using bioorthogonal
N-acetylcysteamine analogs and peptidoglycan O-acetyltransferase B
Yiben Wang, Klare M. Lazor, Kristen Elizabeth DeMeester,
Hai Liang, Tyler K. Heiss, and Catherine Leimkuhler Grimes
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06820 • Publication Date (Web): 12 Sep 2017
Downloaded from http://pubs.acs.org on September 12, 2017
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Post-synthetic modification of bacterial peptidoglycan using
bioorthogonal N-acetylcysteamine analogs and peptidoglycan O-
acetyltransferase B
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Yiben Wang , Klare M. Lazor , Kristen E. DeMeester , Hai Liang, Tyler K. Heiss and Catherine L.
1,2
Grimes *
1
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
2
Department of Biological Chemistry, University of Delaware, Newark, DE 19716, USA
*Correspondence to cgrimes@udel.edu
Supporting Information Placeholder
ABSTRACT: Bacteria have the natural ability to install
protective postꢀsynthetic modifications onto its bacterial
peptidoglycan (PG), the coat woven into bacterial cell wall.
Peptidoglycan Oꢀacetyltransferase B (PatB) catalyzes the
Oꢀacetylation of PG in Gram (–) bacteria, which aids in
bacterial survival, as it prevents autolysins such as lysoꢀ
zyme from cleaving the PG. We explored the mechanistic
details of PatB’s acetylation function and determined that
PatB has substrate specificity for bioorthgonal short Nꢀ
acetyl cysteamine (SNAc) donors. A variety of functionaliꢀ
ty including azides and alkynes were installed on triꢀNꢀ
acetylglucosamine (NAG) , a PG mimic, as well as PG
3
isolated from various Gram (+) and Gram (–) bacterial speꢀ
cies. The bioorthogonal modifications protect the isolated
PG against lysozyme degradation in vitro. We further
demonstrate that this postꢀsynthetic modification of PG can
be extended to use click chemistry to fluorescently label the
mature PG in whole bacterial cells of Bacillus subtilis.
Modifying PG postꢀsynthetically can aid in the developꢀ
ment of antibiotics and immune modulators by expanding
the understanding of how PG is processed by lytic enꢀ
zymes.
Figure 1. A) An acetyl functional group (red) is translocated
across the plasmic membrane by PatA, where it is subsequently
transferred onto the sixth hydroxyl position of NAM by PatB. B)
Unnatural SNAc donors employed to install bioorthogonal
functional groups (blue) onto the peptidoglycan by PatB.
5
gonorrhoeae, leading to resistance of lysozyme degradaꢀ
6
tion. The level of Oꢀacetylation can be as high as 70% in
7
certain bacterial species. Despite the prevalence of Oꢀ
acetylation, the source and delivery mechanism of the aceꢀ
tate is debated; it is hypothesized that the natural source of
the acetate group is from acetylꢀCoA. Since Oꢀacetyl
groups are not found on precursors in the PG biosynthetic
pathway, Oꢀacetylation is considered to occur post synꢀ
Bacterial cell wall, also known as peptidoglycan (PG), is
1
unique to bacteria and is composed of a polymeric netꢀ
8
thetically on either nascent or mature PG. Therefore a
work of alternating units of Nꢀacetylglucosamine (NAG)
and Nꢀacetylmuramic acid (NAM)ꢀpentapeptide carbohyꢀ
mechanism through which the acetate derived from acetylꢀ
CoA is translocated from the cytoplasm to the periplasmic
2
drates that are further crossꢀlinked (Figure 1). PG is a key
9
space via a transporter has been proposed.
component in bacterial cell survival, regulating the osmotic
3
pressure, temperature and pH. PG contributes to the bacteꢀ
The bacterial enzyme, peptidoglycan Oꢀacetyltransferase
B (PatB), was first identified as a PG Oꢀacetyltransferase in
rial cell shape, and serves as the anchoring point for wall
4
10
teichoic acids (WTA) and other extracellular proteins.
Gramꢀnegative bacteria by Clarke and coworkers. PatB is
Bacteria naturally utilize survival mechanisms involving
the postꢀsynthetic modification of its PG. Lysozyme, a
mammalian antimicrobial enzyme, acts on the PG by cleavꢀ
ing the βꢀ1,4ꢀglycosidic bond between NAG and NAM. Oꢀ
acetylation of PG is found in pathogenic bacterial species
including Staphylococcus aureus and Neisseria
proposed to function in conjunction with peptidoglycan Oꢀ
acetyltransferase A (PatA), a transmembrane protein (Figꢀ
ure 1a). Once the acetate is in the periplasm, PatB catalyzes
the transfer of an acetyl group onto the 6ꢀhydroxyl position
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Table 1. Kinetic Data for acetylꢀCoA and SNAc doꢀ
a
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9
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nors Standard deviation of the analysis was from the
average of three independent kinetic experiments.
Acetyltransferase Activity
Entry Donor
pH = 6.5
pH = 8.5
k
cat
=
k
cat
=
0
K
.00544±0.00026
0.422±0.018
1.62±0.17
= 260.2±20.1
1
2
3
5
6
7
8
m
=
m
0.204±0.012
= 26.69±3.74
K
m
=
k
cat /K
k
cat /K
m
k
cat
=
0
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0
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9
0
0.0179±0.0025
0.327±0.030
kcat /K = 54.82±7.16
k
m
=
=
1.62±0.06
1.47±0.23
cat
K
m
=
K
Figure 2. PatB Donors and Acceptors: A) Donors, which retained
the pNP moiety, were synthesized and tested with PatB. B)
Monosaccharide and trisaccharide acceptors tested with PatB. C)
The natural substrate, acetylꢀCoA, Nꢀacetyl cysteamine
derivatives, which contained bioorthgonal handles, were
synthesized and tested to probe for PatB promiscuity.
m
kcat /K = 1,103±91
m
k
cat
=
k
cat
=
0.0370±0.0033
K
0.617±0.071
m
=
m
0.661±0.084
= 55.99±6.58
K
m
=
1.12±0.14
k
cat /K
k
cat /K
m
= 527.1±60.6
kcat
=
0.0288±0.0048
K
k
m
cat
=
=
1.85±0.056
1.69±0.22
11
4
of NAM (Figure 1a). The Clarke group has eloquently
m
=
m
0.531±0.053
= 54.21±6.19
K
demonstrated that PatB accepts paraꢀnitrophenolꢀacetate
k
cat /K
k
m
cat /K = 1,094±93
a
ꢀ1
ꢀ1
ꢀ1
(
pNPꢀAc) (1, Figure 2a), to label small molecule PG mimꢀ
kcat [sec ], K [mM], k /K [M sec ]
m
cat
15
m
ics. In addition, it was demonstrated that PatB hydrolyzes
thioesters (SNAc) (Figure 1b). Moreover, the SNAcꢀ
thioesters would be less susceptible to nonꢀenzymatic hyꢀ
drolysis as compared to the pNP donors. In 2005, Pohl and
coworkers used SNAc analogs to prepare labeled sugar
nucleotides with bioorthogonal functionality, noting that
SNAcs are advantageous compared to their CoA derivaꢀ
tives due to their synthetic simplicity. To assess the utility
of SNAc compounds as PatB donors, Sꢀ(2ꢀacetamidoethyl)ꢀ
ethanethioate (SNAc, 6) , a novel azido SNAc derivative
Sꢀ(2ꢀacetamidoethyl)ꢀ2ꢀazidoethanethioate (SNAz, 7) along
1
2
propionyl CoA. Intrigued by PatB’s promiscuity, we
sought to install bioorthogonal functionality onto the PG,
which could help to illuminate the mechanism and extent of
modification of O-acetylation.
16
The substrate specificity of PatB’s activity was explored
with wild type PatB expressed with Glutathione Sꢀ
17
transferase (GST) and His tags on the Nꢀterminal and Cꢀ
6
17a
terminal, respectively to improve stability and purification
1
3
(
Figure S1). PatB’s esterase activity was measured using
14
an established pNP release assay. The rate of pNP release
from 1 increased in the presence of PatB (Figure S1c). We
found that PatB was able to acetylate a trisaccharide, triꢀN-
with
an
alkynyl
form, 1 Sꢀ(2ꢀacetamidoethyl)ꢀ2ꢀ
7b
alkynylethanethioate (SNAk, 8) were synthesized (Figꢀ
ure 2c). In addition, SNAc donors with extended core strucꢀ
tures (9 and 10) as well as a larger, fluorescent acetyl moieꢀ
ty (SNBodipy, 11) were synthesized to probe the substrate
specificity of PatB. PatB was able to modify (NAG) using
-10 as confirmed by HRLC/MS (Table S1). PatB was unꢀ
acetylꢀglucosamine (NAG) (Figure 2b) (Table S1). This
3
acceptor specificity is in agreement with previous work
where it was demonstrated that PatB requires acceptors that
are trisaccharides or longer and the acetylation of the amino
3
6
12
group at the C2 position is crucial for PatB’s activity.
able to transfer donor 11 onto the (NAG) sugar. Low soluꢀ
3
We next determined if other donors could be utilized. A
variety of acetyl donors, containing alkyne, azido, and pheꢀ
nyl functionalities, were synthesized (2ꢀ4, Figure 2a) and
assayed for the ability to acetylate the model glycan accepꢀ
bility of 9ꢀ11 may attribute to the lower transfer activity as
observed by MS analysis; thus the smaller and more synꢀ
thetically accessible SNAc derivatives 6-8 were chosen as
optimal substrates for PatB. With the (NAG)
fication successfully installed from 8, Alexaꢀ Fluor488 was
alkyne modiꢀ
3
tor (NAG) . High resolution mass spectrometry confirmed
3
1
8
that PatB was able to use 2ꢀ4 to modify (NAG) (Table S1).
successfully attached via click chemistry (Copper cataꢀ
lyzed azideꢀalkyneꢀcycloaddition (CuAAC)) (Figure S3).
3
Kinetic parameters for acetyl transferase were measured by
pNP release (Table S2). The Κ values are in the 0.7ꢀ1.5
m
To measure the catalytic efficiency of PatB with 6-8 in
the presence of (NAG) , a colorimetric assay using
Ellman’s reagent was employed. The k /Κm values for
the SNAc donors were low at pH 6.5 (Table 1). We reaꢀ
soned that pH of the reaction could be important, as the
natural environment (the periplasm of N. gonorrhoeae) in
which PatB resides is slightly basic. At pH 8.5, the cataꢀ
lytic efficiencies of PatB are 10ꢀ20X higher than those obꢀ
served at pH 6.5 (Table 1).
ꢁ
M range and overall k /Κ values were approximately
cat m
3
19
forty times lower for the full length PatB than previously
cat
12
reported for a truncated SUMO tagged PatB. In order to
efficiently install the bioorthogonal functionality using full
length PatB, modified acetyl donors were explored.
20
The proposed natural source of the acetate functional
11
group utilized by PatB is acetylꢀCoA (5, Figure 2c). PatB
modified (NAG) using 5 (Table S1). It was hypothesized
3
that smaller variants of 5 could be used as donors, as a vaꢀ
riety of CoA esterꢀutilizing enzymes are reported to accept
that smaller variants of 5 could be used as donors, as a vaꢀ
riety of CoA esterꢀutilizing enzymes are reported to accept
truncated CoA thioesters known as Nꢀacetylcysteamineꢀ
To evaluate PatB’s acetyltransferase activity on larger,
biologically relevant glycans, we took advantage of its natꢀ
ural ability to prevent the degradation of PG in situ. PG
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use sortase A (SrtA) as a tool for incorporating molecular
was isolated from a bacillus strain with the Oꢀ
acetyltransferase (Oat) gene knockedꢀout (B. sub. ∆Oat).
26a
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handles onto the cell wall of S. aureus and by Liu and
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Figure 3. Lysozyme Degradation Assay: Isolated peptidoglycan
from B. subtilis ꢂOat (A), E. coli (B), P. putida (C) and V.
parahaemolyticus (D) were protected against lysozyme after
modification by PatB and 6 (purple), 7 (black) or 8 (orange) as
compared to the unmodified peptidoglycan (green). Also shown
are blank (blue) and PG without lysozyme (red). Data shown are
averages of three independent reactions. Error bars represent 1
standard deviation of the mean.
Figure 4. SIM and corresponding DIC images of B. subtilis (ꢂOat
and wildꢀtype) cells labeled with AlexaꢀFluor488 (green, top row)
post modification by PatB and 8, PatB and 6, or 8 alone. The cell
wall was also stained with a tetramethylrhodamineꢀWGAꢀ
conjugate (red, second row) to show colocalization of the
fluorophores (yellow, third row). Images are representative of
three biological replicates (Fig. S5 for large fields). Scaleꢀbar = 2
ꢁm.
The peptidoglycan from this mutant strain has been shown
to be devoid of any natural acetylation at the C6ꢀOH posiꢀ
21
Tam to label E. coli cell surfaces through a butelaseꢀ
tion of NAM. The insoluble PG was subjected to lysoꢀ
zyme degradation in the presence and absence of PatB with
1, 6-8; the optical density was measured over time to moniꢀ
26b
mediated ligation method to a transmembrane protein.
B. subtilis ∆Oat and wildꢀtype cells were treated with
PatB and donor 6 or 8. CuAAC was implemented to label
the cells with Alexa Fluor 488ꢀAz and subsequently imꢀ
22
tor the increase or decrease of PG degradation process.
The optical density of unmodified PG decreased over time
with lysozyme treatment, indicating that the PG was being
cut into soluble fragments from lysozyme treatment (Figure
3a, Figure S1d). When the PG was preꢀtreated with PatB
and 1, 6, 7 or 8, then the polymer was protected against
lysozyme degradation (Figure 3a, Figure S1d). Encouraged
by these results, PG was isolated from other bacterial speꢀ
cies Escherichia coli, Vibrio parahaemolyticus, and Pseu-
domonas putida (Supplemental Methods); as these species
27
aged via Structured Illumination Microscopy (SIM).
A
time course determined that optimal labeling was achieved
at 240 min (Figure S4). In both B. subtilis ∆Oat and wildꢀ
type strains, cells displayed higher fluorescence signal inꢀ
tensity when treated with PatB and 8 as compared to treatꢀ
ment with 8 alone or PatB with 6, both of which showed
low background autoꢀfluorescence (Figure 4, Figure S5).
These results indicate that PatB is necessary for efficient
labeling of bacterial cells using SNAc derivatives. Fluoresꢀ
cence in the wildꢀtype B. subtilis cells treated with only 8
could be attributed to OatA’s presence as the fluorescence
is absent when ∆Oat cells are treated with only 8. To verify
that labeling is specific to PG, cells were stained with a
7,23
have not been shown to Oꢀacetylate their PG. PatB conꢀ
ferred lysozyme resistance to the isolated PG from all of
the bacterial strains (Figure 3bꢀd). As expected, some degꢀ
radation of the acetylated PG was observed, as PatB may
not be able to modify every NAM residue in the PG. The
data demonstrate that PatB is capable of modifying small
and large fragments of PG with bioorthogonal functionaliꢀ
ty.
tetramethylrhodamineꢀconjugate of wheat germ agglutinin
28
(
WGA), which binds to NAG. In addition, cultures of B.
21
subtilis ꢂOat were treated with lysozyme (Fig. S6), and a
protective effect for cultures grown in the presence of
PatB/6 was observed, supporting PG acetylation. These
verification approaches were chosen over a MSꢀbased asꢀ
say as the acetylated PG is resistant to lysozyme degradaꢀ
Intrigued by the ability of the bioorthogonal SNAc doꢀ
nors to modify isolated PG, in vivo labeling of Gramꢀ
positive bacterial cells using PatB was pursued. Inspiration
to tag the PG polymer was based on previous strategies
29
24
tion making isolation of detectable fragments extremely
used to label either the peptide stems or carbohydrate
25
difficult. Moreover, the fragile acetyl modifications are
core of PG. While these methods rely on metabolic incorꢀ
poration of unnatural PG building blocks, PatB assisted
labeling would be a complimentary method that relies on
Nature’s ability to postꢀsynthetically modify PG to install
bioorthogonal functionality on the carbohydrate portion of
PG, more specifically the 6ꢀOH of NAM, which to date
remains an unexplored PG labeling target. Similar enzyꢀ
matic approaches have been employed by the Spiegel lab to
30
lost during the harsh PG isolation. The coꢀlocalization of
AlexaꢀFluor488 and tetramethylrhodamineꢀWGA in the
samples treated with PatB and 8 suggest that the labeling
was specific to the PG (Figure 4). The ∆Oat cells have a
higher fluorescence signal as compared to the wildꢀtype,
which can be attributed to more sites available for PatB
modification in ∆Oat cells. Interestingly, the fluorescence
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(
3) (a) Wood, JM. J. Gen. Physiol. 2015, 145, 381. (b) Kremer, L.;
Guerardel, Y.; Gurcha, SS.; Locht, C.; Besra, GS. Microbiol. 2002, 148,
145. (c) Booth, IR. Microbiol. Rev. 1985, 49, 359.
4) Brown, S.; Santa Maria, JP.; Walker, S. Annu. Rev. Microbiol.
signal appears to localize around the septalꢀdivision ring.
Nascent PG is proposed to form at this ring during cell diꢀ
vision, thus it is plausible that PatB is more likely to tarꢀ
get the unmodified PG found in this area. We propose that
the labeling methodology presented here was successful
and specific in labeling PG from B. subtilis cells.
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(
2013, 67, 313.
(5) (a) Ghuysen, M.; Strominger, JL Biochemistry 1963, 2, 1119. (b)
Blundell, JK.; Perkins, HR. J. Bacteriol. 1981, 147, 633.
(
6) Brumfitt, W.; Wardlaw, AC.; Park, JT. Nature 1958, 181, 1783.
(7) Vollmer, W. FEMS Microbiol. Rev. 2008, 32, 287.
8) (a) Gmeiner, J.; Kroll, HP. Eur. J. Biochem. 1981, 117, 171. (b)
The SNAc derivatives employed here shed light on the
mechanism of action of PatA, the proposed acetyl carrier. It
is currently unknown how PatA translocates the acetate
moiety of acetylꢀCoA (Figure 1a). PatB accepts SNAc doꢀ
nors, which contain a thioester bond, suggesting that the
acetate group of acetylꢀCoA is possibly linked to a cysteine
residue of PatA.
(
Dougherty, TJ. J. Bacteriol. 1983, 153, 429. (c) Lear, AL.; Perkins, HR. J
Gen. Microbiol. 1983, 129, 885. (d) Snowden, MA.; Perkins, HR.; Wyke,
AW.; Hayes, MV.; Ward, JB. J. Gen. Microbiol. 1989, 135, 3015.
(9) Moynihan, PJ.; Clarke, AJ. Biochemistry 2014, 53, 6243.
(10) Moynihan, PJ.; Clarke, AJ. J. Biol. Chem. 2010, 285, 13264.
(11) Moynihan, PJ.; Clarke, AJ. Intl. J. of Biochem. & Cell Biol. 2011,
0
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3, 1655.
To conclude, PatB has relaxed substrate specificity for
both pNP and SNAc donors, allowing for the covalent atꢀ
tachment of bioorthogonal functionality directly onto a
small molecule PG fragment mimic, isolated PG from B.
subtilis, E. coli, V. parahaemolyticus, and P. putida, and
intact B. subtilis cells postꢀsynthetically. The modifications
protected the PG against lethal lysozyme degradation mirꢀ
roring Nature’s ability to postꢀsynthetically modify PG. In
addition, wildꢀtype PatB has an optimal catalytic efficiency
at basic pH, which reflects its natural environment. PatB’s
utility to incorporate fluorescent tags via click chemistry on
the PG both in vitro and in whole cells allowed for fluoresꢀ
cent visualization of bacterial cells. PatB proves to be a
useful tool in understanding the acetylation modification of
PG and its resistance mechanisms.
(12) Moynihan, PJ.; Clarke, AJ. J. Biol. Chem. 2014, 289, 16748.
(13) Esposito, D.; Chatterjee, DK. Curr. Opin. Biotechnol. 2006, 17,
353.
(14) Moynihan, PJ.; Clarke, AJ. Anal. Biochem. 2013, 439, 73.
(15) (a) Gerstein, J.; Jencks, WP. JACS 1964, 86, 4655. (b) Jacobsen,
JR.; Hutchinson, CR.; Cane, DE.; Khosla, C. Science 1997, 277, 367. (c)
Mishra, PK.; Drueckhammer, DG. Chemical Reviews 2000, 100, 3283.
(d) Xie, X.; Watanabe, K.; Wojcicki, WA.; Wang, CC.; Tang, Y. Chem. &
Biol. 2006, 13, 1161.
(16) (a) Mizanur, RM.; Jaipuri, FA.; Pohl, NL. JACS 2005, 127, 836.
(17) (a) Schwab, JM.; Klassen, JB. JACS 1984, 106, 7217. (b) Sandy,
M.; Zhu, X.; Rui, Z.; Zhang, W. Org. Lett. 2013, 15, 3396.
(18) Kolb, HC.; Finn, MG.; Sharpless, KB. Angew. Chem. Int. Ed.
Engl. 2001, 40, 2004.
(19) Riddles, PW.; Blakeley, RL.; Zenner, B. Anal. Biochem. 1979, 94,
75.
(20) (a) Brookes, R.; Sikyta, B. Appl. Microbiol. 1967, 15, 224. (b)
Morse, SA.; Hebeler, BH. Infect. Immun. 1978, 21, 87.
(21) Bera, A.; Herbert, S.; Jakob, A.; Vollmer, W.; Gotz, F. Mol. Mi-
crobiol. 2005, 55, 778.
(22) Wang, G.; Lo, L.F.; Forsberg, L.S.; Maier, R. J. Am. Soc. Micro.
2012, 3, e00409.
ASSOCIATED CONTENT
Supporting Information
(23) Clarke, AJ.; Dupont, C. J. Microbiol. 1991, 38, 85.
(24) (a) Sadamoto, R.; Niikura, K.; Sears, PS.; Liu, H.; Wong, C.;
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
including experimental methodology, spectral data, PGꢀisolation
protocols, cellꢀlabeling conditions, kinetics data and synthetic
procedures.
Suksomcheep, A.; Tomita, F.; Monde, K.; Nishimura, S. JACS 2002, 124,
018. (b) Liechti, GW.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, YV.; Vanꢀ
9
Nieuwenhze, M.; Maurelli, AT. Nature 2014, 506, 507. (c) Kuru, E.;
Hughes, HV.; Brown, PJ.; Hall, E.; Tekkam, S.; Cava, F.; de Pedro, MA.;
Brun, YV.; VanNieuwenhze, MS. Angew. Chem. Int. Ed. Engl. 2012, 51,
1
2519. (d) Lebar, MD.; May, JM.; Meeske, AJ.; Leiman, SA.; Lupoli, TJ.;
AUTHOR INFORMATION
Corresponding Author
Tsukamoto, H.; Losick, R.; Rudner, DZ.; Walker, S.; Kahne, D. JACS
2014, 136, 10874. (e) Siegrist, MS.; Whiteside, S.; Jewett, JC.; Aditham,
A.; Cava, F.; Bertozzi, CR. ACS Chem. Biol. 2013, 8, 500. (f) Shieh, P.;
Siegrist, MS.; Cullen. AJ.; Bertozzi, CR. Proc. Natl. Acad. Sci. USA 2014,
*cgrimes@udel.edu
1
11, 5456. (g) Daniel, RA.; Errington, J. Cell 2003, 113, 767. (h) Tiꢀ
yanont, K.; Doan, T.; Lazarus, MB.; Fang, X.; Rudner, D.; Walker, S.
Proc. Natl. Acad. Sci. USA 2006, 103, 11033.
Notes
The authors declare no competing financial interests.
(25) Liang, H.; DeMeester, KE.; Hou, C.; Parent, MA.; Caplan, JL.;
Grimes, CL. Nat. Commun. 2017, 8, 15015.
ACKNOWLEDGMENT
(26) (a) Nelson, JW.; Chamessian, AG.; McEnaney, PJ.; Murelli, RP.;
Kazmiercak, BI.; Spiegel, DA. ACS Chem. Biol. 2010, 5, 1147. (b) Bi, X.;
Yin, J.; Nguyen, G.; Rao, C.; Halim, N.; Hemu, X.; Tam, JP.; Liu, C.
Angew. Chem. Int. Ed. Engl. 2017, 56, 7822.
(27) Gustafsson MG.; Shao, L.; Carlton, PM.; Wang, CJ.; Goluꢀ
bovskaya, IN,; Cande, WZ.; Agard, DA.; Sedat, JW. Biophys. J. 2008, 94,
4957.
C.L.G. is a Pew Biomedical Scholar and Cottrell Scholar, and
thanks the Pew Foundation and the Research Corporation. YW
and KED thank the NIH for support (5T32GM008550). For inꢀ
strumentation support, the Delaware COBRE and INBRE supꢀ
ported this project from NIGMS (5 P30GM110758ꢀ02,
P20GM104316ꢀ01A1 and P20GM103446). We thank Professor
Koh and Wei Bao for their contribution/discussion of SNAc utiliꢀ
ty and Dr. Papa Nii AsareꢀOkai, director of the UD MS facility.
(28) (a) Yoshida, M.; Stadler, J.; Bertholdt, G.; Gerisch, G. EMBO J.
1
984, 3, 2663. (b) Ursell, TS.; Nguyen, J.; Monds, RD.; Colavin, A.;
Billings, G.; Ouzounov, N.; Gitai, Z.; Shaevitz, JW.; Huang, KC. Proc.
Natl. Acad. Sci. USA 2014, 111, E1025.
(29) Davis, KM.; Weiser, JN. Infection and Immunity 2011, 79, 562.
(30) Young, KD. Methods in Microbiol. 1998, 27, 277.
REFERENCES
(31) Weiss, DS. Mol. Microbiol. 2004, 54, 588.
(1) (a) Rogers, HJ. Bacteriol. Rev. 1970, 34, 194. (b) Nanninga, N. Mi-
crobiol. Mol. Biol. Rev. 1998, 62, 110. (c) MenginꢀLecreulx, D.; Lemaitre,
B. J. Endotoxin. Res. 2005, 11, 105.
(32) BissonꢀFilho, AW.; Hsu, Y.; Squyres, GR.; Kuru, E.; Wu, F.;
Jukes, C.; Sun, Y.; Dekker, C.; Holden, S.; VanNieuwenhze, MS.; Brun,
YV.; Garner, EC. Science 2017, 355, 739.
(2) Vollmer, W.; Blanot, D.; de Pedro, MA. FEMS Microbiol. Rev.
2008, 32, 149.
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