Chemical structure and biological activity of a quorum sensing pheromone from Bacillus subtilis subsp. natto

Article abstract of DOI:10.1016/j.bmcl.2015.07.083

Bacillus subtilis subsp. natto secrets a peptide pheromone, named ComX_natto pheromone, as an inducer for biofilm formation containing poly-γ-glutamic acid. Recently, the ComX_natto pheromone was identified to be a hexapeptide with an

Full text of DOI:10.1016/j.bmcl.2015.07.083

Accepted Manuscript
Chemical Structure and Biological Activity of a Quorum Sensing Pheromone
from Bacillus subtilis subsp. natto
Masahiro Okada, Yuta Nakamura, Shunsuke Hayashi, Koki Ozaki, Syohei
Usami
PII:
S0960-894X(15)00787-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.083
BMCL 22972
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
26 June 2015
21 July 2015
24 July 2015
Please cite this article as: Okada, M., Nakamura, Y., Hayashi, S., Ozaki, K., Usami, S., Chemical Structure and
Biological Activity of a Quorum Sensing Pheromone from Bacillus subtilis subsp. natto, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.07.083
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Chemical Structure and Biological Activity of a Quorum Sensing Pheromone from Bacillus
subtilis subsp. natto
a
b
b
b
b
Masahiro Okada *, Yuta Nakamura , Shunsuke Hayashi , Koki Ozaki , Syohei Usami
a
Graduate School of Pharmaceutical Sciences The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo,
113-0033, Japan
b
Graduate School of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai,
Aichi, 487-8501, Japan
Corresponding author.
*
Tel.: +81-3-5841-4741, fax: +81-3-5841-4744, e-mail: okadam@mol.f.u-tokyo.ac.jp
Key words: Bacillus subtilis; natto; posttranslational modification; quorum sensing.
Abstract
Bacillus subtilis subsp. natto secrets a peptide pheromone, named ComXnatto pheromone, as an inducer for
biofilm formation containing poly-γ-glutamic acid. Recently, the ComXnatto pheromone was identified to be a
hexapeptide with an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu, and the tryptophan residue was post-
translationally modified with a farnesyl group. In order to determine the precise modification of the
tryptophan residue, ComXnatto pheromone was synthesized using solid-phase peptide synthesis. Biological
activity of the ComXnatto pheromone was then investigated. It was demonstrated that poly-γ-glutamic acid
production were accelerated by ComXnatto pheromone at more than 1 nM in natto.
1

1
Quorum sensing is a system to activate specific gene expression dependent on cell density. In quorum
sensing, specific pheromones were constantly secreted as an inducer. The pheromone increases with cell
population and triggers gene expression to stimulate various phenomena such as acquisition of virulence,
antibiotic production, biofilm formation, bioluminescence, conjugation, genetic competence, sporulation,
and so on. Since each phenomenon is specifically regulated in response to each quorum sensing pheromone,
quorum sensing is an attractive target for drug discovery. For instance, an inhibitor of quorum sensing
pheromones derived from pathogenic bacteria must be a promising drug candidate as an anti-pathogenic
drug.
Bacillus subtilis secrets ComX pheromone in the control of quorum sensing to stimulate natural genetic
2
competence. ComX pheromone is an oligopeptide synthesized from ComX through posttranslational
modification catalyzed by ComQ and unknown processing system of N-terminal side. In the sufficient
presence of the ComX pheromone, a receptor histidine kinase, ComP, autophosphorylates and donates
phosphate to a response regulator, ComA, via a two-component-system to activate competence gene
3
expression. Striking polymorphism is exhibited in the amino acid sequence of the ComX pheromone
variants in Bacillus strains, but each possesses a modified tryptophan residue at either the 3rd or the 4th
4
residue from the C-terminal. The tryptophan residue is modified with either a geranyl group or a farnesyl
5
group at its γ-position, resulting in the formation of a tricyclic structure.
B. subtilis subsp. natto is well known as a seed of fermented soybeans food in Japan. Natto also possesses
the genes cluster, comQXPAnatto, but the ComXnatto contains a tryptophan residue at neither the 3rd nor the 4th
6
from the C-terminal, but at only the 54th among 73 amino acids. Recently, ComXnatto pheromone was
identified as a hexapeptide having an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu, corresponding to
from the 53th to the 58th residue of ComXnatto, and the 2nd tryptophan residue is posttranslationally modified
7
with a farnesyl group. ComXnatto pheromone is unique in the senses that C-terminal residues of ComXnatto
are processed as well as N-terminal ones in biosynthesis of ComXnatto pheromone and the 5th tryptophan
2

residue from the C-terminal of ComXnatto pheromone is farnesylated. In addition, it was demonstrated that
ComXnatto pheromone stimulated biosynthesis of poly-γ-glutamic acid (γ-PGA) involved in biofilm
formation in natto. Since γ-PGA is water-soluble, edible, and highly sticky polymer utilized to develop
medical applications, ComXnatto pheromone will attract attention as a promising promoter for production of
γ-PGA in natto. Here we report the chemical synthesis of ComXnatto pheromone with solid-phase peptide
synthesis including improvement method for cleavage from the resin to confirm the structure ComXnatto
pheromone. Subsequently, using synthetic ComXnatto pheromone, inducing activity of poly-γ-glutamic acid
production in natto was evaluated.
According to our previous studies, the tryptophan residue of ComXnatto pheromone is probably modified
with a farnesyl group at its γ-position, resulting in the formation of a tricyclic structure in the same manner
5
of ComX168 pheromone and ComXRO-C-2 pheromone. However, it was difficult to purify enough ComXnatto
pheromone for NMR analysis because of instability. In order to confirm the precise chemical structure of
ComXnatto pheromone, we synthesized the modified hexapeptide candidate corresponding to ComXnatto
pheromone. According to the synthesis of ComX pheromones, we synthesized the modified tryptophan
5
,8
residue from Bz-Trp-OMe 1, as shown in Figure 1A. The synthetic protocols of 2-5 were mainly based on
the reference 5c and 8b. Notably, we chose lithium borohydride as a reduction reagent in imine-selective
9
reduction step. We used catecholborane in previous report but it was difficult to purify the product from
5
c, 8b
reaction mixture containing catechol.
After deprotection of the Fmoc group from the amino compounds,
resulting each diastereomeric mixture was easily separated by column chromatography using mixed solvents
of chloroform and methanol to afford optically pure Trp*(Far)-OMe 4. Desired Fmoc-Trp*(Far) 5 for solid-
phase peptide synthesis was easily prepared from Trp*(Far)-OMe 4. Subsequently, Fmoc-Glu(Clt) 7 were
1
0
prepared in 2 steps using commercially available Fmoc-Glu-OAll 6, as shown in Figure 1B. As shown
Figure 1C, peptide bond formation was accomplished with solid-phase peptide synthesis, and then both Clt
resin and the Clt group were cleaved under extremely mild condition by treatment with silica gel in methanol
3

1
1
at 37 °C for 24 h. HPLC purification was carried out to give the synthetic ComXnatto pheromone
1
2
candidate. Other cleavage or purification trials under acidic conditions were failed. For instance, ComXnatto
pheromone was completely decomposed under 1% trifluoroacetic acid (data not shown). It was thus
demonstrated that the improved method by using silica gel in methanol was very effective against cleavage
of Clt resin and deprotection of Clt groups in Fmoc solid phase peptide synthesis.
A
1) NaH, farnesyl bromide,
THF, 0 °C, 5 h, 73%
Far
CO Me
CO Me
2
t-BuOCl, Et N
2
∗
3
Bz-Trp-OMe
N
NH
1
CH
2
Cl
2
, 0 °C,
2) DIBAL, THF, –78 °C,
2 h, 90%
N
H
N
Bz
2
4 h, 96%
3
*(R : S = 0.7 : 1.0)
2
1
) Fmoc-OSu, CH CN,
Far
1) LiOH, THF, CH OH,
H O, rt, 30 min
2
Far
3
3
CO Me
CO H
2
Na CO , H O, rt, 1 h
2
2
3
2
NH
N
2
3
) LiBH , THF, 0 °C, 2 h
2) Fmoc-OSu, CH CN,
3
4
N
N
Fmoc
H
H
H
) piperidine, CH CN, rt,
H
Na CO , H O, rt,
3
2
3
2
3
0 min, 39% (in 3 steps)
1 h, 82% (in 2 steps)
Trp*(Far)-OMe 4
Fmoc-Trp*(Far) 5
B
C
Clt resin
1
) Clt-Cl, Et N,
3
CH Cl , rt,
2
2
SPPS using Fmoc-Glu(Clt) 7, Fmoc-Ile, Fmoc-Pro (× 2),
Fmoc-Trp*(Far), and Fmoc-Lys(Fmoc)
2
h, 79%
Fmoc-Glu-OAll
Fmoc-Glu(Clt)
6
2) Pd(PPh
3
)
4
,
7
Lys-Trp*(Far)-Pro-Pro-Ile-Glu(Clt)-Clt resin
NMA,THF,
silica gel, CH OH, 37 °C, 24 h
0
6
°C, 5 h,
0%
3
NH2
CO H
2
HN
Far = farnesyl
O
H
Clt = 2-chlorotrityl
N
N
N
N
NMA = N-methylanilline
H N
2
N
CO H
2
H
O
O
O
O
ComXnatto pheromone
Figure 1. Synthetic scheme of ComXnatto pheromone.
A, Synthesis of Fmoc-Trp*(Far) 5. The synthetic protocols were mainly based on the reference 5c and 8b.
The synthetic protocol for the reduction by LiBH was described in footnotes 9; B, Synthesis of Fmoc-
4
Glu(Clt) 7. The synthetic protocol was described in footnotes 10; C, Synthesis of ComXnatto pheromone.
We compared the natural ComXnatto pheromone with the synthetic ComXnatto pheromone by LC-MS/MS, as
4

1
1
shown in Figure 2. The synthet ii c ComXnatto peptide exhibited the same retention ti mm e as the natural
pheromone by LC-MS mm onitorin gg at m/z 973.6, as shown Figure 2 and 2b. In add ii tion, the MS/MS
spectrum of the synthetic Com natto pheromone was identical to that of the natural ComXnatto pheromone, as
shown Figure 2c and 2d. Both peptides exhibited a significant signal at m/z 455. 33 (or 455.2) as a y ion of the
a
X
4
fragment ion series of the parent ion at m/z 973.7 (or 973.6). Conseque nn tly, the precise structure of ComXnatto
pheromone was determin ee d to be ident ii cal to the synthetic ComXnatto pheromone.
Figure 2. Analyses of the NN atural ComX
a) EIC m/z 973.6 from LC S analysis for ss ynthetic ComXnatto pheromone; b) EIC m/z 973.7 from LC-MS
analysis for natural ComXnatto pher one; c) MS/MS m/z 973.6 for sy theti cc ComXnatto heromone. An
asterisk represents a pare nn t ion. Each f agment i oo n corresponds to the cc le vage of a pepti dd e bond (b and y
or a farnesyl group (–Far), 845.5 (y ), 769.5 (-Far), 616.4 (b ), 519.6 (b ), 455.3 (y ), 358.1 ( ); d) MS/MS
m/z 973.7 for natural CC om natto pheromone. An asterisk represents a pare nn t ii on. Each fragment ion
corresponds to the cleava gg e f a peptide bond (b and y ) or a farnesyl group (–Far), 845.5 (y ), 769.5 (-Far),
16.4 (b ), 519.6 (b ), 455.3 ( ), 3 55 8.1 (y ).
natto
X PP heromone and the Synthetic ComXnatto pheromone.
-MM
omm
n
p
r
aa
n
n
)
5
3
2
4
y
3
X
oo
n
n
5
6
3
2
y
4
3
5

Successively, we invest ii gated the effect of the ComXnatto pheromone
on γ-PGA production of natto , as
shown in Figure 3. Afte rr the synthetic ComXnatto pheromone (or o tt her sam pp les) was added to a liquid
X
1
2
medium, natto was cultured for 3 days, and the amount of γ-PGA in tt he broth was mm easured. It was
demonstrated that γ-PGA production were a cc celerated by pre-addition of ComXnatto phero mm one at more than
1
nM in contrast to a pl aa in peptide, Lys-Tr pp -P
based on LC-MS analysis exhibited almost same as the synthetic one at 10 nM. Pre-addition of 100 nM of
the synthetic ComXnatto p hh eromone stimulated increase of the yield of γ-PGA more tt han 2 t es compared to
0 μL pre-addition of water as a ne gg ative control. The final concentration oo f the Com natto pheromone in the
culture broth of the negative control was calculated to be 13 nM based on LC-M analysis. Together with
previous genetic studies, these fin dd ings sug est that ComXnatto pheromone pl aa ys a crucial role in γ-PGA
production as a quorum s ee nsing pheromone in natto.
rr o-Pro-Ile-Glu. The natural Co mm Xnatto ph ee romone at 10 nM
imm
1
X
S
g
Figure 3. Effects of Com XX natto pher oo mone on γγ -PGA production of natt oo .
Bars represent the means of triplica tt e samples on γ-PGA production of natto: H
2
O (white), natural ComXnatto
pheromone (red), synthetic ComX
X
natto ph ee romone (blue), and pl aa in pepti dd e Lys-Trp-Pro-Pro-Ile-Glu
6

(yellow). Error bars represent standard deviation.
It is notable that ComQnatto catalyzed farnesylation of the tryptophan residue up to at least the 5th position
from the C-terminal while other known ComQ variants can catalyze isoprenylation from only the 2nd to the
1
3
4
th tryptophan residue from the C-terminal, as previously reported. One possibility is that ComXnatto
possesses a particular sequence. ComX pheromone variants among Bacillus 6 strains possess a modified
tryptophan residue at the 3rd from the C-terminal, except ComXRO-B-2 pheromone that possesses a modified
2
,4
tryptophan residue at the 4th from the C-terminal. Comparing ComXnatto pheromone and ComXRO-B-2
pheromone with the other five ComX pheromone variants, only the two pheromones possess a proline
residue at 2 residue away from the C-terminal side of the modified tryptophan residue. ComQ will probably
receive tryptophan-containing peptides that possess a proline residue on 2 residue away from the C-terminal
side of the tryptophan residue as a substrate, but further studies must be needed to elucidate the details.
Another point to note is that C-terminal residues of ComX were processed as well as N-terminal ones to
produce ComXnatto pheromone. So far, it is not clear which step occurred first, farnesylation or N- or C-
truncation of amino acid residues, but this study has demonstrated that posttranslational farnesylation could
have occurred at the internal tryptophan residue of a substrate in Bacillus species and related bacilli as well
as at that near the C-terminus one.
In conclusion, we synthesized extremely acid-labile ComXnatto pheromone with improved synthetic
methodology in this study to confirm the precise chemical structure of ComXnatto pheromone. In addtion, γ-
PGA production were accelerated in natto by pre-addition of ComXnatto pheromone at more than 1 nM.
ComXnatto pheromone will attract attention as a promising additive for improving the production of γ-PGA
by natto.
Acknowledgments
7

This work was supported by JSPS KAKENHI Grant Numbers 24688011, and MEXT KAKENHI Grant
Numbers 25108724.
References and Notes
1. (a) Camilli, A.; Bassler, B. L. Science 2006, 311, 1113; (b) Bassler, B. L.; Losick, R. Cell 2006, 125, 237;
(c) Williams, P.; Winzer, K.; Chan, W.; Cámara, M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2007, 362,
1
2
3
4
119; (d) Yajima, A. Tetrahedron Lett. 2014, 55, 2773.
. Magnuson, R.; Solomon, J.; Grossman, A. D. Cell 1994, 77, 207.
. Hamoen, L. W.; Venema, G.; Kuipers, O. P. Microbiology 2003, 149, 9.
. (a) Bacon Schneider, K.; Palmer, T. M.; Grossman, A. D. J. Bacteriol. 2002, 184, 410; (b) Ansaldi, M.;
Marolt, D.; Stebe, T.; Mandic-Mulec, I.; Dubnau, D. Mol. Microbiol. 2002, 44, 1561.
5
. (a) Okada, M.; Sato, I.; Cho S. J., Iwata, H.; Nishio, T.; Dubnau, D.; Sakagami, Y. Nat. Chem. Biol. 2005,
, 23; (b) Okada, M.; Yamaguchi, H.; Sato, I.; Tsuji, F.; Qi, J.; Dubnau, D.; Sakagami, Y. Biosci. Biotechnol.
1
Biochem. 2007, 71, 1807; (c) Okada, M.; Yamaguchi, H.; Sato, I.; Tsuji, F.; Dubnau, D.; Sakagami, Y.
Biosci. Biotechnol. Biochem. 2008, 72, 914;(d) Okada, M. Biosci Biotechnol Biochem. 2011, 75, 1413.
6. (a) Tran, L.-S. P.; Nagai, T.; Itoh, Y. Mol. Microbiol. 2000, 37, 1159; (b) Nishito, Y.; Osana, Y.; Hachiya,
T.; Popendorf, K.; Toyoda, A.; Fujiyama, A.; Itaya, M.; Sakakibara, Y. BMC Genomics 2010, 11, 243.
7. Hayashi, S.; Usami, S.; Nakamura, Y.; Ozaki, K.; Okada, M. Biosci Biotechnol Biochem. 2015, in press.
8. (a) Okada, M.; Sato, I.; Cho, S. J.; Suzuki, Y.; Ojika, M.; Dubnau, D.; Sakagami, Y. Biosci. Biotechnol.
Biochem. 2004, 68, 2374; (b) Okada, M.; Sato, I.; Cho, S. J.; Dubnau, D.; Sakagami, Y. Tetrahedron 2006,
2, 8907; (c) Okada, M.; Yamaguchi, H.; Sato, I.; Cho, S. J.; Dubnau, D.; Sakagami, Y. Bioorg. Med. Chem.
6
Lett. 2007, 17, 1705; (d) Tsuji, F.; Kobayashi, K.; Okada, M.; Yamaguchi, H.; Ojika, M.; Sakagami, Y.
Bioorg. Med. Chem. Lett. 2011, 21, 4041.
9. A typical protocol is follows: To a solution of the imine in THF (0.1 M) was added 0.5 equivalent of
8

lithium borohydride at 0 °C. After stirring for 2 h, the reaction mixture was poured into 0.1 M phosphate
buffer (pH 7.0). The mixture was extracted with EtOAc and the organic layer was washed with saturated
2 4
aqueous NaCl, dried over Na SO , and evaporated. The residue was purified by silica gel column
chromatography to give diasteromers of Fmoc-Trp*(Far)-OMe. Since the compound was unstable, so they
were carried on to the next step immediately.
1
0. A typical protocol is follows: To a solution of Fmoc-Glu-OAll 6 and 2.5 equivalent of N-
ethyldiisopropylamine (DIPEA) in CH Cl (0.1 M) at room temperature under nitrogen was added 1.1
equivalent of 2-chlorotrityl chloride (Clt-Cl). After the reaction mixture was stirred for 2 h, the reaction
mixture was washed with saturated aqueous NaCl, dried over Na SO , and evaporated. The residue was
2
2
2
4
purified by silica gel column chromatography eluted with 0.5% DIPEA containing solvent mixture to
prevent from Clt deprotection, to give Clt ester of Fmoc-Glu-OAll [Fmoc-Glu(Clt)-OAll] in 79% overall
yield. Since the Clt ester was unstable, they were carried on to the next step immediately. To a solution of
Fmoc-Glu(Clt)-OAll and 10 equivalent of NMA in THF (0.1 M) at 0 °C under nitrogen was added 0.01
equivalent of Pd(PPh
3
)
4
. After the reaction mixture was stirred at 0 °C for 5 h, the organic layer was washed
and saturated aqueous NaCl, and dried over Na SO . After evaporation of the
O to give Fmoc-Glu(Clt) 7 as a pale yellow powder in
with 5% aqueous KHSO
4
2
4
3 2
solution, the residue was recrystallized from CHCl /Et
1
6
1
0% overall yield. H-NMR (400 MHz, CDCl
3
): 7.76 (m, 2H), 7.59 (m, 2H), 7.40-7.20 (m, 18H), 5.48 (br,
1
3
H), 4.50-4.40 (m, 3H), 4.23 (m, 1H), 2.72 (m, 1H), 2.34 (m, 1H), 1.28 (m, 1H), 0.90 (m, 1H). C-NMR
(
3
100 MHz, CDCl ): 171.4, 170.6, 155.8, 143.7, 143.5, 142.3, 141.1, 139.4, 133.5, 131.6 131.3, 131.2, 129.1,
1
1
27.8, 127.6, 127.4, 126.9, 125.8, 125.0, 119.8, 119.0, 90.2, 67.0, 66.1, 53.4, 47.1, 30.9, 27.5. The H and
1
3
C NMR spectra can be found in Supplementary Information.
1
9
1
1. HPLC conditions were as follows: X-select CSH C18 column (Waters) with linear gradient from 60% to
8% methanol in 10 mM of aqueous ammonium formate.
2. After the sample was added to 10 mL of liquid medium, natto was statically incubated in the medium for
9

72 h at 37 °C. After sodium chloride (585 mg, 10 mmol) was added, the broth was centrifuged and filtrated.
After 20 mL of ethanol was added to the filtrate, the suspension was centrifuged to collect precipitate. The
pellet was resolved in water, and the solution was filtrated through an ultrafiltration membrane (100 kDa) to
afford almost pure γ-PGA as a filtration residue. After the freeze-drying, the weight of γ-PGA was measured.
1
H NMR (D
2
O, t-BuOH: δ 1.23): δ4.15 (m, 1H; relative), 2.34 (m, 2H; relative), 2.05 (m, 1H; relative), 1.91
1
(
m, 1H; relative). The H NMR spectrum can be found in Supplementary Information.
13. (a) Tsuji, F.; Ishihara, A.; Kurata, K.; Nakagawa, A.; Okada, M.; Kitamura, S.; Kanamaru, K.; Masuda,
Y.; Murakami, K.; Irie, K.; Sakagami, Y. FEBS Lett. 2012, 586, 174; (b) Tsuji, F.; Ishihara, A.; Nakagawa,
A.; Okada, M.; Kitamura, S.; Kanamaru, K.; Masuda, Y.; Murakami, K.; Irie, K.; Sakagami, Y. Biosci.
Biotechnol. Biochem. 2012, 76, 1492; (c) Okada, M.; Ishihara, A.; Yamasaki, R.; Tsuji, F.; Hayashi, S.;
Usami, S.; Sakagami, Y. Biosci. Biotechnol. Biochem. 2014, 78, 550.
10

Legends for Figures.
Figure 1. Synthetic scheme of ComXnatto pheromone.
A, Synthesis of Fmoc-Trp*(Far) 5. The synthetic protocols were mainly based on the reference 5c and 8b.
4
The synthetic protocol for the reduction by LiBH was described in footnotes 9; B, Synthesis of Fmoc-
Glu(Clt) 7. The synthetic protocol was described in footnotes 10; C, Synthesis of ComXnatto pheromone.
Figure 2. Analyses of the Natural ComXnatto Pheromone and the Synthetic ComXnatto pheromone.
a) EIC m/z 973.6 from LC-MS analysis for synthetic ComXnatto pheromone; b) EIC m/z 973.7 from LC-MS
analysis for natural ComXnatto pheromone; c) MS/MS m/z 973.6 for synthetic ComXnatto pheromone. An
asterisk represents a parent ion. Each fragment ion corresponds to the cleavage of a peptide bond (b
or a farnesyl group (–Far), 845.5 (y ), 769.5 (-Far), 616.4 (b ), 519.6 (b ), 455.3 (y ), 358.1 (y ); d) MS/MS
m/z 973.7 for natural ComXnatto pheromone. An asterisk represents a parent ion. Each fragment ion
corresponds to the cleavage of a peptide bond (b and y ) or a farnesyl group (–Far), 845.5 (y ), 769.5 (-Far),
16.4 (b ), 519.6 (b ), 455.3 (y ), 358.1 (y ).
n n
and y )
5
3
2
4
3
n
n
5
6
3
2
4
3
Figure 3. Effects of ComXnatto pheromone on γ-PGA production of natto.
Bars represent the means of triplicate samples on γ-PGA production of natto: H
2
O (white), natural ComXnatto
pheromone (red), synthetic ComXnatto pheromone (blue), and plain peptide Lys-Trp-Pro-Pro-Ile-Glu
yellow). Error bars represent standard deviation.
(
11

Graphical Abstract
Chemical Structure and Biological Activity
of a Quorum Sensing Pheromone from
Bacillus subtilis subsp. natto
Masahiro Okada*, Yuta Nakamura, Shunsuke Hayashi, Koki Ozaki, Syohei Usami
12