One Enzyme to Build Them All: Ring-Size Engineered Siderophores Inhibit the Swarming Motility of Vibrio

Article abstract of DOI:10.1021/acschembio.8b00084

Bacteria compete for ferric iron by producing siderophores, and some microbes engage in piracy by scavenging siderophores of their competitors. The macrocyclic hydroxamate siderophore avaroferrin of Shewanella algae inhibits swarming of Vibrio alginolyticus by evading this piracy. Avaroferrin, as well as related putrebactin and bisucaberin, are produced by the IucC-like synthetases AvbD, PubC, and BibC^C. Here, we have established that they are capable of synthesizing not only their native product but also other siderophores. Exploiting this relaxed substrate specificity by synthetic precursors generated 15 different ring-size engineered macrocycles ranging from 18- to 28-membered rings, indicating unprecedented biosynthetic flexibility of the enzymes. Two of the novel siderophores could be obtained in larger quantities by precursor-directed biosynthesis in S. algae. Both inhibited swarming motility of Vibrio and, similar to avaroferrin, the most active one exhibited a heterodimeric architecture. Our results demonstrate the impact of minor structural changes on biological activity, which may trigger the evolution of siderophore diversity.

Full text of DOI:10.1021/acschembio.8b00084

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One Enzyme to Build Them All – Ring Size Engineered
Siderophores Inhibit Swarming Motility of Vibrio
Sina Rütschlin, Sandra Gunesch, and Thomas Boettcher
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00084 • Publication Date (Web): 13 Apr 2018
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One Enzyme to Build Them All – Ring Size Engineered Sider-
ophores Inhibit Swarming Motility of Vibrio
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Sina Rütschlin, Sandra Gunesch and Thomas Böttcher*
#Department of Chemistry, Konstanz Research School Chemical Biology, Zukunftskolleg, University of Konstanz,
78457 Konstanz, Germany.
*Email: thomas.boettcher@uni-konstanz.de
Supporting Information Placeholder
rioxamine B is even in medicinal use as drug for treating iron
ABSTRACT: Bacteria compete for ferric iron by producing
siderophores and some microbes engage in piracy by scav-
overload in humans.7 While many siderophores are synthe-
sized by nonribosomal peptide synthetases (NRPS), the elu-
cidation of the biosynthesis of desferrioxamines in Strepto-
myces has led to the discovery of a novel class of NRPS-
independent siderophore (NIS) synthetases.8-10 Also dimeric
macrocyclic siderophores are produced via some NIS synthe-
tases, whereby these enzymes catalyze two separate steps:
the initial dimerization of the substrates and the following
macrocyclization reaction (Figure 1A).9, 11
enging siderophores of their competitors. The macrocyclic
hydroxamate siderophore avaroferrin of Shewanella algae
inhibits swarming of Vibrio alginolyticus by evading this
piracy. Avaroferrin as well as related putrebactin and bi-
sucaberin are produced by the IucC-like synthetases AvbD,
PubC, and BibCC. Here, we established that they are capable
of synthesizing not only their native product but also other
siderophores. Exploiting this relaxed substrate specificity by
synthetic precursors generated 15 different ring size engi-
neered macrocycles ranging from 18- to 28-membered rings,
indicating unprecedented biosynthetic flexibility of the en-
zymes. Two of the novel siderophores could be obtained in
larger quantities by precursor-directed biosynthesis in S.
algae. Both inhibited swarming motility of Vibrio and similar
to avaroferrin the most active one exhibited a heterodimeric
architecture. Our results demonstrate the impact of minor
structural changes on biological activity, which may trigger
the evolution of siderophore diversity.
Availability of the essential element iron is one of the most
important factors limiting bacterial growth. In response to
low solubility of ferric iron in the environment, bacteria have
evolved to produce and secrete a wealth of small molecule
chelators, called siderophores, with extremely high affinity
for ferric iron.1 The resulting siderophore-iron complexes are
taken up by the cells via specific receptors. Since sidero-
phores bind iron outside of the cells and the cell that profits
from the iron uptake is not necessarily the same cell that
produced the siderophore, they may present public goods.2, 3
This makes siderophores susceptible to social cheating by
bacteria that lack the costly pathways for siderophore bio-
synthesis but encode the corresponding uptake receptor – a
phenomenon which is also known as siderophore piracy.2, 4
The enormous competition for iron is consequently spurring
the continuous evolution of new siderophores along with
highly specific receptors. Not only are siderophores im-
portant for microbial interactions but they also play major
roles as virulence factors of pathogenic bacteria in human
and animal diseases.5, 6 The hydroxamate siderophore desfer-
Figure 1. A) Steps in the biosynthesis of dimeric macrocyclic
siderophores catalyzed by some IucC-like type C NIS synthe-
tases. Substrate molecules HS[X]A are first dimerized and
finally macrocyclized to a symmetric or asymmetric sidero-
phore [X+X’]. B) Structures of the three siderophores pro-
duced via AvbD in S. algae. C) Phylogenetic tree of IucC-like
type C NIS synthetases with RhbC (type A NIS) of Sinorrhi-
zobium meliloti serving as outgroup. Bootstrap values >70%
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are indicated at the corresponding branches. The scale bar
represents amino acid substitutions per site.
ed the synthetic and native precursors with AvbD, PubC and
BibCC in ATP reaction buffer. Based on the amounts of mac-
rocycles produced by each enzyme, we calculated the relative
ratios (Supporting Information). All three enzymes were able
to convert the artificial precursors alone or in combination
with the native substrates to the corresponding homo- and
heterodimeric macrocycles (Figure 2, Table S1).
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We have previously discovered that the chimeric cyclic hy-
droxamate siderophore avaroferrin of Shewanella algae B516
inhibits swarming of Vibrio alginolyticus and concluded that
it does so by blocking siderophore piracy of Vibrio.12 In con-
trast, the homodimeric counterparts putrebactin and bi-
sucaberin were considerably less active swarming inhibitors.
We recently investigated the biosynthesis of these three
related siderophores which are all produced by the same
Shewanella algae B516 strain and differ only by the length of
their diamine building blocks (Figure 1B).13
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We demonstrated that AvbD, a IucC-like type C NIS synthe-
tase is responsible for the dimerization and macrocyclization
of the monomeric precursors. AvbD showed a much higher
affinity and catalytic efficiency for the larger cadaverine
derived substrate HS[5]A leading to bisucaberin in compari-
son to the shorter putrescine derived HS[4]A, the precursor
of homodimeric putrebactin. Yet, the cell’s substrate pool
appeared to be adjusted to maximize production of the ad-
vantageous chimeric avaroferrin.13
Stimulated by these results, we were now interested to also
investigate the specificity of the related dimerization-
macrocyclisation type C synthetases BibCC and PubC, which
were originally described to be the corresponding homo-
logues for the biosynthesis of bisucaberin and putrebactin,
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respectively.11, To this aim, we first performed a detailed
phylogenetic analysis of the type C NIS synthetases by max-
imum likelihood (Figure 1C, Figure S1).
In the resulting tree, PubC, AvbD, and BibCC homologues
clustered in three separate groups, which together formed a
sister clade to DesD and AlcC homologues, the biosynthesis
enzymes of the trimeric siderophore desferoxamine and the
dimeric hydroxylated siderophore alcaligin, respectively.
AvbD was hereby more closely related to BibCC as compared
to PubC homologues. The phylogenetic distance of PubC,
AvbD, and BibCC raised the question if these differences
would be also reflected in substrate discrimination of the
enzymes towards the synthesis of their designated native
siderophore products.
We thus synthesized the two native biosynthetic precursors
HS[4]A and HS[5]A as described previously.11, 14, 15 We then
heterologously expressed PubC, AvbD, and BibCC in E. coli
and purified the enzymes to reconstitute the ATP-dependent
enzyme reaction in vitro. Surprisingly, each of the enzymes
was capable of producing all three siderophores, putrebactin,
avaroferrin, and bisucaberin without major differences in
substrate discrimination. We conclude, that in species using
PubC and BibCC the available substrate pool of a cell dictates
the siderophore ratios as we have demonstrated previously
for AvbD in S. algae B516.13 Accordingly, pure putrebactin
producers lack the AvbA/BibA homologue of a lysine decar-
boxylase for cadaverine production and strains producing
mainly bisucaberin supposedly have a bias towards generat-
ing the cadaverine-derived precursor. As rather substrate
availability than specificity of the synthetase determines the
production of the corresponding native siderophores by
these IucC-like enzymes, we suspected that the enzymes
might also accept other non-native substrates. To investigate
this possibility, we synthesized precursor molecules HS[3]A
to HS[6]A, and HS[8]A with different chain lengths in the
diamine subunit (Supporting information). We then convert-
Figure 2. Macrocycle [X+X’] production by A) AvbD, B)
BibCC and C) PubC given in relative amounts formed after 4
h with 4 µM enzyme (A) and (C) or 6 h and 8 µM enzyme (B)
for conversion of combinations of substrates HS[X]A at 15°C
for AvbD and PubC and at 30°C for BibCC. The number of
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methylene-groups in the chain between the nitrogen atoms
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is given on the-x axis.
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All homodimeric products and heterodimeric combinatorial
products could be detected with ring sizes ranging from 18-
to 38-membered macrocycles, which were identified based
on high-resolution mass spectra (Table S1). Furthermore, all
compounds were confirmed by fragmentation analysis with
MS-MS (Figure S2). All three enzymes were relatively similar
in accepting a broad range of non-natural substrates. PubC
was slightly more capable of incorporating short-chain pre-
cursors than AvbD and BibCC displayed the narrowest sub-
strate range (Figure 2, Table S1). Surprisingly, macrocycle
production was controlled on the level of substrate discrimi-
nation and not by the ring size of the product.
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While for example [3+8] was produced only in traces, [5+6]
was formed in considerable quantities although both com-
prise a 23-membered ring. Also combinations of HS[3]A with
other precursors yielded only small amounts of the hetero-
dimeric products. While the native substrates HS[4]A and
HS[5]A were generally preferred, non-natural HS[6]A was
surprisingly well accepted by the enzymes and PubC yielded
[6+6] and [5+6] even as the most abundant products. The
heterodimeric [5+6] also was one of the major products of
AvbD and BibCC. Our results thus indicate a general highly
relaxed substrate selectivity for three homologous IucC-like
synthetases, which may allow for rapid evolution of new
siderophores. To test if the new macrocylic compounds also
behaved as siderophores, we supplemented the samples with
10 mM aqueous solution of FeCl3 to observe mass shifts to
the respective iron-complexes (Table S2).16 For almost all
artificial macrocycles produced, with the exception of [3+3]
and [8+3], we could detect the corresponding 1:1 iron-
siderophore complexes [M+Fe]+ with their characteristic
isotopic pattern, indicating formation of a tetradentate iron
chelate. We also investigated chelation of other ions, includ-
ing Ga3+, vanadate, and molybdate, which were all three
found to form 1:1 complexes with the majority of macrocycles
(Table S3).
Figure 3. Production of siderophores by live cells of S. algae
fed with hexane-1,6-diamine. A) Structures of newly pro-
duced siderophores. B) LC-MS trace of siderophore produc-
tion of precursor-directed biosynthesis by Shewanella algae
fed with 1,6-diaminohexane. EICs (Extracted Ion Chromato-
grams) are shown for putrebactin m/z = 373.20 [M+H]+,
avaroferrin, m/z = 387.20 [M+H]+, bisucaberin and [4+6] m/z
= 401.22 [M+H]+, [5+6] m/z = 415.23 [M+H]+ and [6+6] m/z =
429.25 [M+H] +.
Two of the artificial siderophores [5+6] and [6+6] could be
extracted in sufficient amounts and isolated. The products
were purified to homogeneity by HPLC and the structures
were characterized by 1D and 2D NMR spectroscopy and
mass spectrometry (Figures S4-S15). Previously reported
attempts of feeding artificial diamines of different chain
lengths to another Shewanella strain did not result in prod-
uct formation,17 indicating that our strain S. algae B516 has an
exceptionally relaxed substrate acceptance also in AvbB and
AvbC, which are responsible for the HS[X]A precursor bio-
synthesis. We were now interested if the isolated new sider-
ophores had effects on swarming inhibition of Vibrio. Previ-
ous work has demonstrated that swarming of Vibrio algino-
lyticus is dependent on availability of ferric iron and inhibi-
tion of swarming is mediated by iron-chelation. However,
swarming inhibition did not correlate with the binding affini-
ties for iron of the corresponding chelators. We reasoned
that not the strength of iron chelation but rather the ability
of V. alginolyticus to utilize the chelated iron determines the
activity of swarming inhibitors.12 The genome of V. alginolyt-
icus encodes numerous siderophore importers for xenosider-
ophores, which it does not produce by itself and related
Vibrio species are known to engage in siderophore piracy, the
poaching of siderophores from competitors.18, 19 We had thus
postulated that in contrast to siderophores like desferriox-
amine B that does not inhibit swarming, heterodimeric
avaroferrin of S. algae thwarts piracy by Vibrio and cutting
off Vibrio from its ferric iron resource halts its swarming
motility.12 We now reasoned, that non-natural siderophores
like [5+6] and [6+6] may also escape siderophore piracy and
thereby inhibit swarming of V. alginolyticus. To investigate
this possibility, we performed a previously established
swarming assay on carrageenan plates with the prolific
swarming strain V. alginolyticus B522, which had been co-
Having confirmed that most of the macrocyclic products
were indeed siderophores, we speculated that some of these
non-natural siderophores like native avaroferrin may also
prevent Vibrio’s siderophore piracy and thereby inhibit
swarming motility. However, as the reconstituted in vitro
reactions did not yield sufficient amounts for further experi-
ments, we explored the possibility of using the live cells of S.
algae as bioreactors for precursor-directed siderophore bio-
synthesis. Cells were grown on carrageenan plates under iron
starvation conditions and native putrescine (required for
HS[4]A) synthesis was suppressed using an ornithine decar-
boxylase inhibitor.16 Feeding of different diamines was fol-
lowed by solvent extraction and LC-MS analysis of the ex-
tracts. These resulted only for the artificial substrate hexane-
1,6-diamine in a considerable degree of product formation.
Shewanella algae produced significant amounts of the non-
natural engineered siderophores [5+6], [4+6] and [6+6] next
to the natural siderophores putrebactin [4+4], avaroferrin
[4+5] and bisucaberin [5+5] (Figure 3, Figure S3, Table S4).
isolated with avaroferrin producing S. algae B516 from the
20
same specimen of marine macroalga.12,
Along with the
native controls avaroferrin and bisucaberin previously isolat-
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ed,13 the purified engineered siderophores were added on
heterodimeric architecture of avaroferrin was considerably
more active than the homodimeric [6+6] siderophore akin to
bisucaberin. It is thus conceivable, that the asymmetric
structure of heterodimers is less well recognized by Vibrio.
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paper discs surrounding a swarming colony of V. alginolyti-
cus and we monitored for the formation of a clear inhibition
zone (Figure 4A). None of the compounds had antibacterial
activities and also did not inhibit Escherichia coli and Bacil-
lus subtilis up to 200 µM.
The fierce battle for scavenging ferric iron and thwarting
siderophore piracy of their competitors has triggered the
evolution of an enormous structural diversity of microbial
siderophores.21, 22 A common theme in the evolution of sider-
ophores is the acquisition and mutation of modular genes
tailoring and modifying the siderophore’s structure.23-25 In
other cases different independent gene clusters shuffled into
one organism coordinate the production of novel sidero-
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phores as reported for rhodochelin or serratiochelins.4,
Diversity can also be generated by multispecificity of en-
zymes in the biosynthesis incorporating multiple different
substrates.27 We recently demonstrated that this is the case
for AvbD in S. algae whereby product ratios are dictated by
the available substrate pool.13 Our new results suggest that
this concept is generally applicable to an entire subgroup of
IucC-like enzymes. PubC and BibCC which had been previ-
ously investigated in context of their native products putre-
bactin and bisucaberin,11, 14, 28 do not drastically differ in their
specificity from AvbD. All of them can be thus regarded to be
broad-spectrum dimerization and macrocyclization enzymes.
Figure 4. Swarming inhibition by ring-size engineered sider-
ophores. A) Swarming assay with [5+6] and [6+6] in compar-
ison to avaroferrin [4+5] and bisucaberin [5+5] at 50 nmol
showing inhibition zones imaged at different time points. B)
Time-resolved development of the clear zone as distance to
the swarming front measured from the inoculated paper
discs. C) Concentration dependent series of distance to front
(zone diameter) after 8 hours.
While previous attempts to generate artificial dimeric mac-
rocyclic siderophores by precursor-directed biosynthesis
successfully led to unsaturated versions of putrebactin,16 we
report here for the first time ring-size engineered variants
ranging from 18- to 28-membered rings. This extraordinarily
broad spectrum of macrocycles of different size and architec-
ture that can be produced simply by feeding different precur-
sors indicates an unprecedented potential of these enzymes
in the evolution of structural diversity of siderophores. An
important example of how already small changes to a sidero-
phore can have major ecological or even pathophysiological
consequences is illustrated by pathogenic strains of E. coli
and Salmonella. These strains glycosylate and linearize the
siderophore enterobactin, which allows them to evade sabo-
tage of iron scavenging by the human immune response.29
Accordingly, our results with artificial ring-size variants of
dimeric hydroxamate siderophores demonstrate that minor
changes to a siderophore core scaffold such variation of ring-
size or heterodimeric versus homodimeric architecture can
have significant effects on competing species, which here is
illustrated by the inhibition of swarming Vibrio.
A first test revealed that the engineered siderophores [5+6]
and [6+6] exhibited considerable swarming inhibition activi-
ty (Figure 4A). To test whether the engineered siderophores
inhibited swarming as result of limiting bioavailablitiy of
ferric iron, we added excess of 2-fold ferric iron onto the
paper discs inoculated with the active siderophores (Figure
S16). The inhibitory effect was reversed by addition of ferric
iron confirming that [5+6] inhibited by the same mode of
action as previously established for avaroferrin. The pᴍ(FeIII)
values of the new siderophores were comparable to those of
putrebactin, avaroferrin, and bisucaberin, confirming again
that not the binding affinities but the ability of V. alginolyti-
cus to use the siderophore-bound iron are critical for swarm-
ing inhibition (Figure S17). To quantitate the effect of the
engineered artificial siderophores in respect to avaroferrin,
we monitored swarming inhibition in dependence of concen-
tration and time. As shown in Figure 4A and B the size of
clear swarming-inhibition zones [6+6] and [5+6] were con-
siderably more persistent and larger compared to bisucaber-
in [5+5] whereby [5+6] was more active than [6+6]. Over the
entire concentration range [5+6] was about as active as
avaroferrin (Figure 4B).
METHODS
Additional methods can be found in the Supporting Infor-
mation.
Synthesis of N-hydroxy-N-succinyl-alkylenediamines.
The corresponding HS[X]A substrates were produced from
dihaloalkanes using Gabriel synthesis followed by substitu-
tion of the second halogen with tert-butyl N-
(benzyloxy)carbamate, partial de- and reprotection and
coupling with succinic anhydride, according to a method
described previously.14
These results demonstrate that our ring-size engineered
artificial siderophores are able to mimic the effect of native
avaroferrin to inhibit swarming Vibrio by depriving the cells
of ferric iron, which is required for swarming motility. It has
been previously proposed that only those siderophores,
which cannot be readily scavenged by V. alginolyticus are
active swarm inhibitors.12 It could thus be expected that
swarming Vibrio is less likely capable of utilizing artificial
ring size engineered siderophores, which we could now con-
firm. Interestingly, the [5+6] siderophore, resembling the
Overexpression and purification of AvbD, BibCC and
PubC. The proteins were recombinantly expressed with N-
terminal Strep-tag in E. coli. Proteins were purified from
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StrepTrap™ HP affinity column (GE Healthcare Bio-Sciences
AB) on an ÄKTA™ start protein purification system.
ACKNOWLEDGMENT
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We thank A. Marx and his group for their generous support
and the Zukunftskolleg of the University of Konstanz. We
thank N. Willassen and H. Hansen, UiT the Artic University
of Norway for providing a genome sample of A. salmonicida
LFI1238. We especially thank M. Prothiwa, H. Bußkamp, M.
Mex and D. Hammler for their help with mass spectrometry.
Enzyme reactions. Substrates HS[X]A were incubated in 1:1
ratio (total 2 mM) with 4 mM ATP, 15 mM MgCl2, 25 mM
Tris-HCl (pH 8.0) and 4 μM enzyme (AvbD and PubC) in a
final volume of 140 μL (or 70 µL for BibCC) for four hours at
15°C. Reactions with BibCC were carried out at 30 °C with
8 μM enzyme for 6 h. In aliquots of 40 µL, reactions were
stopped by addition of 3 µL 10% trichloroacetic acid. LC-MS
analysis of the reaction mixture was carried out as described
in the Supporting Information.
REFERENCES
9
(1) Miethke, M., and Marahiel, M. A. (2007) Siderophoreꢀbased iron
acquisition and pathogen control, Microbiol Mol Biol Rev 71, 413ꢀ451.
(2) Cordero, O. X., Ventouras, L. A., DeLong, E. F., and Polz, M. F.
(2012) Public good dynamics drive evolution of iron acquisition strategies
in natural bacterioplankton populations, Proc Natl Acad Sci U S A 109,
20059ꢀ20064.
(3) West, S. A., Griffin, A. S., Gardner, A., and Diggle, S. P. (2006)
Social evolution theory for microorganisms, Nat Rev Microbiol 4, 597ꢀ
607.
(4) Seyedsayamdost, M. R., Cleto, S., Carr, G., Vlamakis, H., Joao
Vieira, M., Kolter, R., and Clardy, J. (2012) Mixing and matching
siderophore clusters: structure and biosynthesis of serratiochelins from
Serratia sp. V4, J Am Chem Soc 134, 13550ꢀ13553.
(5) Takase, H., Nitanai, H., Hoshino, K., and Otani, T. (2000) Impact
of siderophore production on Pseudomonas aeruginosa infections in
immunosuppressed mice, Infect Immun 68, 1834ꢀ1839.
(6) Hirschmann, M., Grundmann, F., and Bode, H. B. (2017)
Identification and occurrence of the hydroxamate siderophores aerobactin,
putrebactin, avaroferrin and ochrobactin C as virulence factors from
entomopathogenic bacteria, Environ Microbiol 19, 4080ꢀ4090.
(7) Fisher, S. A., Brunskill, S. J., Doree, C., Gooding, S., Chowdhury,
O., and Roberts, D. J. (2013) Desferrioxamine mesylate for managing
transfusional iron overload in people with transfusionꢀdependent
thalassaemia, Cochrane Database Syst Rev, CD004450.
(8) BaronaꢀGomez, F., Wong, U., Giannakopulos, A. E., Derrick, P.
J., and Challis, G. L. (2004) Identification of a cluster of genes that directs
desferrioxamine biosynthesis in Streptomyces coelicolor M145, J Am
Chem Soc 126, 16282ꢀ16283.
(9) Kadi, N., OvesꢀCostales, D., BaronaꢀGomez, F., and Challis, G. L.
(2007) A new family of ATPꢀdependent oligomerizationꢀmacrocyclization
biocatalysts, Nat Chem Biol 3, 652ꢀ656.
(10) Challis, G. L. (2005) A widely distributed bacterial pathway for
siderophore biosynthesis independent of nonribosomal peptide
synthetases, Chembiochem 6, 601ꢀ611.
(11) Kadi, N., Arbache, S., Song, L., OvesꢀCostales, D., and Challis,
G. L. (2008) Identification of a gene cluster that directs putrebactin
biosynthesis in Shewanella species: PubC catalyzes cyclodimerization of
NꢀhydroxyꢀNꢀsuccinylputrescine, J Am Chem Soc 130, 10458ꢀ10459.
(12) Böttcher, T., and Clardy, J. (2014) A Chimeric Siderophore Halts
Swarming Vibrio, Angew Chem Int Ed Engl 53, 3510ꢀ3513.
(13) Rütschlin, S., Gunesch, S., and Böttcher, T. (2017) One Enzyme,
Three Metabolites: Shewanella algae Controls Siderophore Production via
the Cellular Substrate Pool, Cell Chem Biol 24, 598ꢀ604 e510.
(14) Kadi, N., Song, L., and Challis, G. L. (2008) Bisucaberin
biosynthesis: an adenylating domain of the BibC multiꢀenzyme catalyzes
cyclodimerization of NꢀhydroxyꢀNꢀsuccinylcadaverine, Chem Commun
(Camb) 0, 5119ꢀ5121.
(15) Lifa, T., Tieu, W., Hocking, R. K., and Codd, R. (2015) Forward
and reverse (retro) iron(III) or gallium(III) desferrioxamine E and ringꢀ
expanded analogues prepared using metalꢀtemplated synthesis from endoꢀ
hydroxamic acid monomers, Inorg Chem 54, 3573ꢀ3583.
(16) Soe, C. Z., and Codd, R. (2014) Unsaturated macrocyclic
dihydroxamic acid siderophores produced by Shewanella putrefaciens
using precursorꢀdirected biosynthesis, ACS Chem Biol 9, 945ꢀ956.
(17) Soe, C. Z., Telfer, T. J., Levina, A., Lay, P. A., and Codd, R.
(2016) Simultaneous biosynthesis of putrebactin, avaroferrin and
bisucaberin by Shewanella putrefaciens and characterisation of complexes
with iron(III), molybdenum(VI) or chromium(V), J Inorg Biochem 162,
207ꢀ215.
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Precursor-directed biosynthesis and isolation of sider-
ophores. S. algae B516 was grown on carrageenan NBE plates
for 4 to 5 days at 30°C with 100 μM 2,2’-bipyridyl as iron
chelator, 10 mM 1,4-diamino-2-butanone dihydrochloride as
ornithine decarboxylase inhibitor and 10 mM 1,6-
diaminohexane. The agar was soaked overnight in ethyl
acetate followed by isopropanol. The isopropanol extract was
evaporated and the mixture was pre-fractionated on a Sep-
Pak SPE C18 cartridge (Waters) and subsequently purified by
multiple preparative HPLC runs as detailed in the Supporting
Information.
Vibrio swarming assays. Standard swarming assays were
performed on carrageenan NBE plates, prepared from 1.5 %
(w/v) carrageenan for gel preparation in Nutrient Broth E. A
6 mm blank paper disc (BD BBL) was placed in the center of
a plate surrounded by four further blank paper discs with 2
cm distance between the centers of each disc. The central
disc was inoculated with 5 μL overnight culture of V. algino-
lyticus B522 in NBE medium and incubated at 30°C. After 12
h the surrounding discs were inoculated with 5 μL of a
10 mM DMSO stock of siderophores.
ASSOCIATED CONTENT
Supporting Information
Syntheses, characterization of siderophores, mass spectra
and bioassays. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Email: thomas.boettcher@uni-konstanz.de (T.B.)
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
Notes
The authors declare no competing financial interests.
Funding Sources
We gratefully acknowledge funding by the Emmy Noether
program of the Deutsche Forschungsgemeinschaft (DFG), EU
FP7 Marie Curie Zukunftskolleg Incoming Fellowship Pro-
gram – University of Konstanz grant no. 291784, the Fonds
der Chemischen Industrie (FCI), the Konstanz Research
School Chemical Biology (KoRS-CB), and SFB969 (DFG). SR
was supported by a KoRS-CB fellowship.
(18) Kim, C. M., Park, Y. J., and Shin, S. H. (2007) A widespread
deferoxamineꢀmediated ironꢀuptake system in Vibrio vulnificus, J Infect
Dis 196, 1537ꢀ1545.
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(19) Tanabe, T., Funahashi, T., Miyamoto, K., Tsujibo, H., and
(26) Bosello, M., Robbel, L., Linne, U., Xie, X., and Marahiel, M. A.
(2011) Biosynthesis of the siderophore rhodochelin requires the
coordinated expression of three independent gene clusters in Rhodococcus
jostii RHA1, J Am Chem Soc 133, 4587ꢀ4595.
Yamamoto, S. (2011) Identification of Genes, desR and desA, Required
for Utilization of Desferrioxamine B as a Xenosiderophore in Vibrio
furnissii, Biol Pharm Bull 34, 570ꢀ574.
1
2
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4
5
6
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9
(20) Böttcher, T., Elliott, H. L., and Clardy, J. (2016) Dynamics of
Snakeꢀlike Swarming Behavior of Vibrio alginolyticus, Biophys J 110,
981ꢀ992.
(21) Lee, W., van Baalen, M., and Jansen, V. A. (2012) An
evolutionary mechanism for diversity in siderophoreꢀproducing bacteria,
Ecol Lett 15, 119ꢀ125.
(27) Wuest, W. M., Sattely, E. S., and Walsh, C. T. (2009) Three
siderophores from one bacterial enzymatic assembly line, J Am Chem Soc
131, 5056ꢀ5057.
(28) OvesꢀCostales, D., Kadi, N., and Challis, G. L. (2009) The longꢀ
overlooked enzymology of a nonribosomal peptide synthetaseꢀ
independent pathway for virulenceꢀconferring siderophore biosynthesis,
Chem Commun (Camb), 6530ꢀ6541.
(29) Fischbach, M. A., Lin, H. N., Liu, D. R., and Walsh, C. T. (2006)
How pathogenic bacteria evade mammalian sabotage in the battle for iron,
Nat Chem Biol 2, 132ꢀ138.
(22) Hider, R. C., and Kong, X. (2010) Chemistry and biology of
siderophores, Nat Prod Rep 27, 637ꢀ657.
(23) Fischbach, M. A., Walsh, C. T., and Clardy, J. (2008) The
evolution of gene collectives: How natural selection drives chemical
innovation, Proc Natl Acad Sci U S A 105, 4601ꢀ4608.
(24) Franke, J., Ishida, K., and Hertweck, C. (2014) Evolution of
siderophore pathways in human pathogenic bacteria, J Am Chem Soc 136,
5599ꢀ5602.
(25) Crosa, J. H., and Walsh, C. T. (2002) Genetics and assembly line
enzymology of siderophore biosynthesis in bacteria, Microbiol Mol Biol
Rev 66, 223ꢀ249.
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TOC graphic
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