Hybrid biosynthesis of roseobacticides from algal and bacterial precursor molecules

Article abstract of DOI:10.1021/ja508782y

Roseobacticides regulate the symbiotic relationship between a marine bacterium (Phaeobacter inhibens) and a marine microalga (Emiliania huxleyi). This relationship can be mutualistic, when the algal host provides food for the bacteria and the bacteria produce growth hormones and antibiotics for the algae, or parasitic, when the algae senesce and release p-coumaric acid. The released p-coumaric acid causes the bacteria to synthesize roseobacticides, which are nM-μM toxins for the algae. We examined the biosynthesis of roseobacticides and report that all roseobacticide precursors play critical roles during the mutualist phase of the symbiosis. Roseobacticides are biosynthesized from the algal growth promoter, the major food molecule provided by the algal cells, and the algal senescence signal that initiates the mutualist-to-parasite switch. Thus, molecules that are beneficial during mutualism are diverted to the synthesis of toxins during parasitism. A plausible mechanism for assembling roseobacticides from these molecules is proposed.

Full text of DOI:10.1021/ja508782y

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Communication
Hybrid Biosynthesis of Roseobacticides from
Algal and Bacterial Precursor Molecules
Mohammad R. Seyedsayamdost, Rurun Wang, Roberto Kolter, and Jon Clardy
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja508782y • Publication Date (Web): 08 Oct 2014
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Hybrid Biosynthesis of Roseobacticides from Algal and Bacterial Pre-
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†
,*
†
‡
§,*
Mohammad R. Seyedsayamdost, Rurun Wang, Roberto Kolter, and Jon Clardy
†
Department of Chemistry, Princeton University, Princeton, New Jersey 08544
‡
§
Department of Microbiology & Immunobiology and Department of Biological Chemistry & Molecular Pharmacology,
Harvard Medical School, Boston, Massachusetts 02115
Supporting Information Placeholder
ber of vitamins, notably vitamin B12, have also been implicat-
ABSTRACT: Roseobacticides regulate the symbiotic
relationship between a marine bacterium (Phaeobacter inhibens)
and a marine microalga (Emiliania huxleyi). This relationship can
be mutualistic, when the algal host provides food for the bacteria
and the bacteria produce growth hormones and antibiotics for the
algae, or parasitic, when the algae senesce and release p-coumaric
acid. The released p-coumaric acid causes the bacteria to synthe-
size roseobacticides, which are nM-M toxins for the algae. We
examined the biosynthesis of roseobacticides and report that all
roseobacticide precursors play critical roles during the mutualist
phase of the symbiosis. Roseobacticides are biosynthesized from
the algal growth promoter, the major food molecule provided by
the algal cells, and the algal senescence signal that initiates the
mutualist-to-parasite switch. Thus, molecules that are beneficial
during mutualism are diverted to the synthesis of toxins during
parasitism. A plausible mechanism for assembling roseobacticides
from these molecules is proposed.
1
5,16
ed.
This mutually-beneficial relationship changes in the pres-
ence p-coumaric acid (pCA, 4), a potential senescence signal pro-
3
duced by the algal host. In response to pCA, the bacteria activate
an otherwise silent biosynthetic pathway that generates the
algaecidal roseobacticides (5–7). While the small molecules in-
volved in this symbiosis have been identified, the biosynthetic
pathways that produce them remain unknown.
P. inhibens
O
O
S
O
O
OH
COOH
HO
DMSP
(1)
R
S
O
pCA
(4)
Roseobacticides
S
Tropodithietic
acid (TDA, 2)
R=
OH
S
OH
O
N
H
Phenylacetic
acid (3)
OH
A (5)
B (6)
C (7)
Naturally-occurring small molecules have motivated important
chemical studies on structure, synthesis, and biosynthesis, provid-
ed therapeutic agents for medicine, and supplied reagents for bi-
E. huxleyi
1
,2
ology. While relatively few studies focused on their natural
roles, those that did have also revealed interesting chemical and
ecological insights. As part of a larger project on the natural roles
of bacterially produced small molecules, especially those involved
in regulating symbiotic interactions, we uncovered the
roseobacticides, which are produced by the marine bacteria
Figure 1. Model for metabolite exchange in the algal-bacterial
symbiosis between E. huxleyi and P. inhibens. The symbiotic
interaction comprises two phases, a mutualistic phase (green ar-
rows) and a parasitic phase (red arrows). In the mutualistic phase,
E. huxleyi provides the C- and S-source dimethylsulfonio-
propionate (1). The bacteria in return produce TDA (2), an antibi-
otic that protects the host from bacterial pathogens, and
phenylacetic acid (3), an algal growth promoter. In the parasitic
phase, P. inhibens generates the algaecidal roseobacticides (5-7)
in response to p-coumaric acid (4), a likely senescence signal
produced by E. huxleyi.
3
,4
Phaeobacter inhibens DSM 17395 and P. inhibens 2.10. The
producers belong to the diverse bacterial roseobacter clade and
help regulate the intermittent symbiosis with Emiliania huxleyi,
3,5-7
a microscopic single-celled alga that forms massive blooms,
synthesizes elaborate calcium carbonate shields, and removes
significant quantities of carbon dioxide from the environment
8
,9
through photosynthesis. This report identifies the biosynthetic
precursors for the roseobacticides, which turn out to be hybrid
molecules composed of fragments contributed by both the algae
and the bacteria.
The recent discovery of eleven roseobacticide analogs suggest-
ed that they may be generated from tropone, an aromatic amino
4
acid (Phe, Tyr, or Trp), and MeSH (in the case of analogs A-C).
To examine these hypotheses, we initiated a series of isotope
feeding experiments coupled with multi-dimensional NMR and
high-resolution MS to uncover the precursors of roseobacticides.
Elegant studies by Schulz and colleagues recently showed that P.
inhibens can convert Phe into tropone or tropone hydrate (i.e. 5-
Recent studies by several research groups have shown that
roseobacters engage in a dynamic and opportunistic symbiosis
3
,10,11
with microalgal cells.
The algae provide food in the form of
dimethylsulfoniopropionate (Fig. 1, DMSP, 1), which P. inhibens
1
7
cycloheptene-1,3-dione). As shown in Scheme 1, these com-
pounds are generated with different (effective) planes of sym-
can use as a source of sulfur and carbon. In return, the bacteria
12-14
produce tropodithietic acid (TDA, 2),
a broad-spectrum anti-
1
3
2
metry. Using 1,2- C -phenylacetic acid (8) as a substrate, the
biotic that protects against pathogenic bacteria, and phenylacetic
acid (3), an algal growth hormone. In certain interactions, a num-
1
3
pathway would provide 2- C-tropone (9), which is asymmetrical-
ly-labeled and would be expected to give two isotopomers of
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1
3
2
13
roseobacticide B. In contrast, 1,2- C
2
-phenylacetic acid yields 5-
Interestingly, neither H nor C from phenylacetic acid were
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cycloheptene-1,3-dione labeled at the unique C2 position (10) and
would thus result in roseobacticide B with a single isotopomer.
incorporated into the aromatic substituent of roseobacticide B. A
2
similar result was obtained with H
5
-indoleacetic acid indicating
that the side chain is not derived from phenylacetic acid or
indoleacetic acid, in the cases of roseobacticides B and C, respec-
tively. To illuminate the source of the aromatic side chain,
Scheme 1. Expected patterns of isotope incorporation into
1
3
2
roseobacticide B from 1,2- C- and ring- H
acid.
5
-phenylacetic
13
deuterated and C-containing amino acids were employed. P.
2
inhibens DSM 17395 cultures were grown in the presence of H
5
-
1
Trp and roseobacticide C isolated and characterized. The H-
NMR spectrum in Fig. 3A & 3B clearly show a disappearance of
2
the indole ring protons consistent with incorporation of H
5
-indole
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
into roseobacticide C. These results are further confirmed with
8
+
exp
+
HR-MS data, which yield [M+H]
313.1063 ([M+H]
calc
3
H
13.1059). Analogous experiments were carried out with ring-
10
2
13
2
4
-Tyr, 2- C-Tyr, and ring- H
5
-Phe. The Tyr analogs revealed
plane of
symmetry
2
incorporation of the H
4
-phenol into roseobacticide A as well as
placement of the α-carbon of Tyr as the carbonyl-C2 of
roseobacticide A (Fig. 3C, Scheme 2A, Fig. S2). Experiments
2
with ring- H -Phe gave three distinct labeling patterns, containing
5
11
12
13
either 2, 5, or 7 deuterons (Fig. 3D & Scheme 2B). NMR and
HR-MS analyses revealed that these consisted of either two deu-
To distinguish between these options, P. inhibens DSM 17395
terons in the tropone moiety, five in the phenyl group, or the
1
3
2
7
was cultured in YTSS media supplemented with 0.4 mM 1,2- C
2
-
combination of these two to yield H -roseobacticide B (Fig. S3).
phenylacetic acid. Production of roseobacticides was induced by
addition of sinapic acid and roseobacticide B subsequently isolat-
These results establish the role of Phe as the source of both the
tropone moiety and the aromatic side chain in roseobacticide B, as
well as those of Tyr and Trp in generating the other roseobacticide
analogs.
4
ed as previously described and assessed by HR-MS and 2D-
+
NMR. The former gave [M+H] expt 270.0673 consistent with in-
1
3
+
calc
13
corporation of a single C ([M+H]
270.0670, Table S1). C-
8
NMR, gHSQC, and gHMBC spectra demonstrated labeling at a
single position, the C8 of roseobacticide B, entirely consistent
with 5-cycloheptene-1,3-dione, but not tropone, serving as a pre-
17
6
16
5
4
15
cursor for roseobacticide B (Fig. 2, Fig. S1). These results were
12
16
2
further corroborated using ring- H
5
-phenylacetic acid (11). Thiel
14
4
et al. have reported that 11 is converted to 12 and 13, again
12
2
demonstrating an asymmetric distribution of H in tropone versus
14
17
6
5
symmetric labeling in the 5-cycloheptene-1,3-dione, in which
15
8
solvent exchange leads to loss of deuterons α to the carbonyl
A
B
1
7
groups. Isolation of roseobacticide B from production cultures
grown in the presence of 11 yielded an isotopomer containing two
+
+
deuterons ([M+H] calc 271.0762, [M+H] expt 271.0745). These
results indicate that phenylacetic acid, a growth stimulant pro-
duced during the beneficial phase of the symbiosis, is employed
as a precursor for toxin production in the parasitic phase.
7.6
7.4
7.2
7.0
6.8
6.6
289.0839
269.0640
271.0764
285.0589
1
59 Hz
5
4
12
1
3
8
286.0623
1
.9 Hz
274.0953
14
6
285.0587
276.1078
105
110
115
120
6
1
2
5
4
1
3
C
D
E
1
4
m/z
m/z
m/z
8
Figure 3. Aromatic amino acids and signal pCA serve as
1
roseobacticide precursors. (A) Assigned H NMR spectrum of
1
roseobacticide C. (B) H spectrum of roseobacticide C isolated
2
from cultures containing ring- H -Trp, showing incorporation of
5
125
2
5
H -indole. (C) HR-MS spectrum of roseobacticide A isolated
7
.6
7.4
7.2
7.0
6.8
6.6
2
2
from cultures containing ring- H -Tyr. Both protonated and H -
4
4
phenol-bearing isotopomers are observed. (D) HR-MS spectrum
Figure 2. Incorporation of an algal growth promoter into
roseobacticide B. Shown is the gHSQC spectrum of 5 isolated
from P. inhibens cultures containing compound 8. A single corre-
2
5
of roseobacticide B isolated from cultures containing ring- H -
Phe. Three deuterated isotopomers, and the all-protonated form
are observed. (E) HR-MS spectrum of roseobacticide A isolated
13
lation is observed corresponding to C insertion at C8 position of
13
from cultures containing 2- C-pCA showing incorporation of
roseobacticide B. As expected, a C-H coupling constant of 159
Hz is observed at C8. Labeled roseobacticide B is shown along
signal pCA into roseobacticide A. See Scheme 2 and Table S1.
1
with the assigned shifts in the H trace.
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4,25
Given that amino acids serve as roseobacticide precursors, we
containing compounds, some with highly unusual structures.
In a natural context, DMSP (1, Fig. 1) is likely the only sulfur
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
wondered whether this knowledge could be used to generate new
analogs and whether pCA, which could be converted to Tyr via
2
6
source, which can be converted to Cys. To examine a role for
Cys in the production of roseobacticides, P. inhibens DSM 17395
cultures were supplemented with S-Cys. HR-MS data convinc-
ingly show incorporation of S into roseobacticides B & E re-
vealing that the sulfur atoms in the thiomethyl and methyl disul-
fide substituents are provided by Cys (Scheme 2F, Fig. S6). Thus,
these results suggest that DMSP, the food molecule provided by
the algal host, is utilized by the bacteria as a substrate to generate
the algal toxin.
1
8
the enzyme tyrosine ammonia lyase, might be incorporated into
roseobacticide A. This incorporation would represent an unusual
scenario in which the algal signal that activates the roseobacticide
biosynthetic pathway, also serves as a substrate for its synthesis.
We tested these hypotheses by performing production cultures
3
4
27
3
4
2
13
with ring- H -pCA, 2- C-pCA, and 3-fluorotyrosine. The latter
4
was commercially available and cultures containing 3-F-Tyr in-
deed gave rise to fluorinated roseobacticides A and D, as demon-
1
19
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
strated by H, F, and gCOSY NMR spectra, and by HR-MS
studies (Scheme 2C, Fig. S4, Tables S1 & S2). These results
highlight the flexibility of the enzymes in the roseobacticide bio-
synthetic pathway that can accommodate a fluorine substituent on
the Tyr substrate. To test whether signal pCA might be incorpo-
Scheme 2. Observed patterns of isotope incorporation for
a number of labeled precursor molecules.
2
13
rated, ring- H -pCA and 2- C-pCA were synthesized by a
a
4
A
Knoevenagal condensation of the appropriately labeled malonic
1
9
acid and 4-hydroxybenzaldehyde isotopomers (see SI). Surpris-
ingly, NMR and HR-MS analysis of roseobacticide A and D
2
2
clearly showed incorporation of the H
4
-phenol group of ring- H -
4
13
pCA into the side chain as well as insertion of the 2- C in pCA as
B
1
3
the 2- C carbonyl in roseobacticide A (Fig. 3E, Scheme 2D, Fig.
S5). In the natural system, local concentrations of pCA are not
known as the bacterial cells adhere to an algal cell. In our algal-
free system, pCA signaling and pCA-incorporation into
3
,4
roseobacticides both occur at the same concentration, >0.4 mM.
C
Collectively, the data above indicate that pCA serves both as a
trigger and a substrate for roseobacticides, which contain a phenol
group at position C3, and that the bacteria incorporate the algal
signal pCA into the algal toxin.
A number of plausible mechanisms may be proposed for the in-
corporation of aromatic amino acids into roseobacticides. Incor-
poration of the α-carbon of Tyr as the C2-carbonyl of
roseobacticide A suggests that the Tyr carboxyl group is lost dur-
ing the course of biosynthesis (Scheme 2A). The absence of the
amino group necessitates a deaminated analog as the direct pre-
cursor. Accordingly, we tested incorporation of several products
of the Phe/Tyr degradation pathway. One such product is the
deaminated phenylpyruvic acid, generated from Phe by a putative
a
D
E
aminotransferase (PGA1_c29420), which can be found in the
2
F
genome of P. inhibens DSM 17395. Ring- H -phenylpyruvic acid
5
2
0
was synthesized enzymatically as previously described. Inclu-
sion of this compound in production cultures of P. inhibens DSM
2
1
7395 clearly demonstrated incorporation of the H
5
-phenyl group
a
2
13
Experiments with
4
H - and C-labeled precursors were carried out separately in
reactions A and D, but are combined above for brevity.
into roseobacticide B (Scheme 2E, Table S1). Two other poten-
2
tial substrates, ring- H -phenylglycine, obtained commercially,
5
2
and ring- H -phenylglyoxylic acid, synthesized by SeO -mediated
Scheme 3. Proposed pathway for production of phen-
ylglyoxylyl-CoA in P. inhibens DSM 17395
5
2
2
21
a
5
oxidation of the corresponding ring- H -phenylacetic acid, both
failed to incorporate into roseobacticides. The isotope feeding
results regarding the side chain of roseobacticides may best be
explained by the pathway in Scheme 3. In this pathway, Phe is
a
b
c
converted to phenylpyruvate by an aminotransferase. Ior1
16,22
(PGA1_c04490), recently identified,
then catalyzes conver-
a
a: PGA1_c29420, b: PGA1_c04490, c: PGA1_c28900
sion of phenylpyruvate to phenylacetyl-CoA. Thus phenylacetic
acid is not an intermediate in this pathway perhaps explaining the
lack of incorporation into the side chain of roseobacticides.
BLAST searches against the genome of P. inhibens DSM 17395
also revealed an enzyme complex that has been shown to generate
phenylglyoxylyl-CoA from phenylacetyl-CoA (PGA1_c28900)
suggesting that either may serve as the direct precursor to the
The results from the isotope feeding data above may be inte-
grated to propose a biosynthetic pathway for roseobacticides (Fig.
). Three amino acids, Tyr, Phe and Cys, are the precursors to
4
roseobacticide A. Phe provides phenylacetic acid, and Cys, de-
rived from the food molecule DMSP, affords the sulfur atoms(s)
(Fig. 4A). Tyr provides the side chain as well as part of the 5-
membered ring lactone, and may be derived from Phe or from
pCA, a conversion catalyzed by the enzyme Tyr ammonia lyase
(TAL). BLAST searches in P. inhibens DSM 17395 reveal an
enzyme with high homology to the E. coli TAL, putatively anno-
tated as a His ammonia lyase (PGA1_c36340). In roseobacticides
2
3
roseobacticides (see below).
The experiments above have focused on the source of the sev-
en-membered ring and the side chain in roseobacticides. We next
addressed the source of the thiomethyl group in 5–7. P. inhibens
has been shown to synthesize a number of small, volatile sulfur-
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B and C, Tyr is replaced with Phe and Trp, respectively. In our
AUTHOR INFORMATION
Corresponding Authors
mrseyed@princeton.edu
jon_clardy@hms.harvard.edu
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
current working model (Fig. 4B), a 5-cycloheptene-1,3-dione
carbanion attacks the α-ketone carbonyl of an aromatic glyoxylyl-
CoA (15) to give 16. Lactone formation is driven by the loss of
CoA forming 17. DMSP then, most likely via Cys, provides the
sulfur for the thiomethyl group by an unknown mechanism, per-
haps akin to that recently proposed for sulfur insertion into
Notes
27
TDA. Additional experiments will be necessary to test this pro-
The authors declare no competing financial interests.
posed pathway, and studies addressing the genetics and enzymol-
ogy of roseobacticide biosynthesis are currently underway.
ACKNOWLEDGMENT
S
O
34
We thank Prof. Jeroen Dickschat for the kind gift of S-Cys, Dr.
Istvan Pelczer at the Princeton Department of Chemistry NMR
facility for assistance with NMR data acquisition, and grants from
the National Institutes of Health (GM098299 to M.R.S.,
GM086258 to J.C. and GM82137 to R.K.) for support of this
work.
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
OH
S
OH
A
OH
3
O
O
4
1
O
HO
OH
Phe
Phe
Base:
H
Base:
SCoA
OH
O
B
R=H
REFERENCES
O
O
OH
H
CoAS
3 O
a
R
15
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O
O
O
B
14
16
A
R=OH
CoAS
H2O
Phe
O
Tyr
pCA
c
b
O
(5) Geng, H.; Belas, R. Curr. Opin. Biotechnol. 2010, 21, 332.
O
O
(6) Wagner-Döbler, I.; Biebl, H. Annu. Rev. Microbiol. 2006, 60,
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R
R
2
55.
17
S
O
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Figure 4. Precursors and proposed pathway for roseobacticide
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algal molecules (1 & 4) and from the algal growth promoter (3)
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(
9) Holligan, P. M.; Fernandez, E.; Aiken, J.; Balch, W. M.; Boyd, P.;
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Tyr may be provided by
a
putative TAL
Microbiol. 2014, 5, 311.
(
PGA1_c36340, reaction b) or from Phe (reaction c). See text for
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In conclusion, the roseobacticides are assembled from three
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Phenylacetic acid (3) is a bacterially-produced algal growth pro-
moter, p-coumaric acid (4) is an algal senescence molecule, and
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roseobacticides. Thus, beneficial molecules in the mutualistic
phase are converted into toxins in the parasitic phase – a remarka-
ble example of metabolic economy. This economy likely reflects
both the nutrient-poor environment in which the symbiosis occurs
and the necessity for a switch-like conversion. Finally, the study
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set of chemical reactions, but can also provide insights into the
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ASSOCIATED CONTENT
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2
13
2
of ring- H -pCA, 2- C-pCA, ring- H -phenylpyruvic acid, and
4
5
2
ring- H
5
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13
bacticides isolated from cultures containing ring- H -Tyr, 2- C-
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