Biosynthesis of amphi-enterobactin siderophores by Vibrio harveyi BAA-1116: Identification of a bifunctional nonribosomal peptide synthetase condensation domain

Article abstract of DOI:10.1021/ja5019942

The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar

Full text of DOI:10.1021/ja5019942

Communication
pubs.acs.org/JACS
Biosynthesis of Amphi-enterobactin Siderophores by Vibrio harveyi
BAA-1116: Identification of a Bifunctional Nonribosomal Peptide
Synthetase Condensation Domain
Hannah K. Zane,† Hiroaki Naka,‡ Federico Rosconi,§ Moriah Sandy,† Margo G. Haygood,‡
and Alison Butler*,†
†Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106-9510, United States
‡Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon 97239-3098, United States
§Departamento de Bioquímica y Genom
́ ́
ica Microbianas, Instituto de Investigaciones Biologicas Clemente Estable, Montevideo
11600, Uruguay
S
* Supporting Information
Enterobactin, the lactone of tris-2,3-dihydroxybenzoyl-L-serine,
is the prototypic catecholate siderophore produced by many
bacteria, most notably Escherichia coli8 (Figure 1C). An
encoded long-chain fatty acid CoA ligase (FACL) is present
in the V. harveyi genome near the putative enterobactin
biosynthetic gene cluster (Figure 1B). This enzyme is predicted
to be similar to enzymes that are involved in the acylation of
peptides, often located in the vicinity of the NRPS cluster.9−11
The organization of the V. harveyi gene cluster, specifically
the FACL nearby the NRPS genes, in conjunction with the
continued discovery of new N-terminally acylated peptide
siderophores,11,12 led us to hypothesize that V. harveyi produces
acylated enterobactin-like siderophores. We report herein the
structural characterization of the amphi-enterobactin side-
rophores produced by V. harveyi ATCC BAA-1116, in addition
to the biosynthetic genes, designated as the aeb gene cluster
(Figure 1B, Table S1). Through heterologous expression and in
vitro reconstitution of amphi-enterobactin biosynthesis, we
reveal unique dual activity of the single condensation domain of
the NRPS protein, AebF.
V. harveyi ATCC BAA-1116 was grown in a low-iron
synthetic seawater medium. After 2 days, the culture was
harvested by centrifugation, the bacterial cell pellet was
extracted with methanol, and the extract was purified with
solid-phase adsorption and RP-HPLC (λ = 310 nm for catechol
absorption). Eight peaks gave a positive CAS assay response for
iron binding, consistent with the presence of apo-siderophores
(Figure 2).13 ESI-MS of these compounds showed a range in
mass from m/z 927 to m/z 987 (Table S2, Figures S1−S8).
Enterobactin (m/z 670 [M+H]+) was not detected in either the
culture supernatant or the bacterial cell pellet.
ABSTRACT: The genome of Vibrio harveyi BAA-1116
contains a nonribosomal peptide synthetase (NRPS) gene
cluster (aebA−F) resembling that for enterobactin, yet
enterobactin is not produced. A gene predicted to encode
a long-chain fatty acid CoA ligase (FACL), similar to
enzymes involved in the biosynthesis of acyl peptides,
resides 15 kb away from the putative enterobactin-like
biosynthetic gene cluster (aebG). The proximity of this
FACL gene to the enterobactin-like synthetase suggested
that V. harveyi may produce amphiphilic enterobactin-like
siderophores. Extraction of the bacterial cell pellet of V.
harveyi led to the isolation and structure determination of a
suite of eight amphi-enterobactin siderophores composed
of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine
and acyl-L-serine. The FACL knockout mutant, ΔaebG V.
harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi,
do not produce amphi-enterobactins. The amphi-entero-
bactin biosynthetic machinery was heterologously ex-
pressed in Escherichia coli and reconstituted in vitro,
demonstrating the condensation domain of AebF has
unique activity, catalyzing two distinct condensation
reactions.
Vibrio harveyi BAA-1116 (recently reclassified as V. campbel-
lii1,2) is widely used to study quorum sensing, due to its
quorum-regulated bioluminescence.3,4 Quorum sensing in V.
harveyi is known to regulate siderophore production, but the
identity of the siderophore had not been determined.3
Siderophores are low-molecular-weight iron(III) chelators
that solubilize and transport iron(III) into the cell.5 Side-
rophores contribute to competitiveness in environmental
bacteria and serve as virulence factors in pathogens, including
Vibrios.6 Microbial colonization of an animal host depends
critically on the ability to obtain sufficient iron, yet is also
challenged by the low levels of available iron in the host
environment.7
ESI-MS/MS of the eight compounds produced a similar
pattern, with three major daughter ions at m/z 670, m/z 447,
and m/z 224 ([M+H]+) (Figure S4A), as observed in the ESI-
MS/MS of enterobactin.14 However, in each spectrum,
additional fragment ions were observed, varying in mass
among the compounds, analogous in pattern to other
siderophore suites that are N-terminally acylated with differing
The genome of V. harveyi strain ATCC BAA-1116 contains a
nonribosomal peptide synthetase (NRPS) gene cluster
resembling that for enterobactin biosynthesis (Figure 1, Table
S1), yet we did not detect enterobactin in cultures of V. harveyi.
Received: February 26, 2014
Published: April 4, 2014
© 2014 American Chemical Society
5615
dx.doi.org/10.1021/ja5019942 | J. Am. Chem. Soc. 2014, 136, 5615−5618

Journal of the American Chemical Society
Communication
Figure 1. (A) Schematic of the genes composing the enterobactin biosynthetic cluster in E. coli. (B) Schematic of the genes composing the putative
amphi-enterobactin cluster in V. harveyi: yellow, FACL; blue, DHBA biosynthetic proteins; green, NRPS proteins; red, P-pant transferase (see Table
S1 for details). Boxes with dashed lines indicate the genetic regions deleted in this study. (C) Enterobactin. (D) Amphi-enterobactin C12-OH (1;
1
peak 4 in Figure 2) depicted with H−1H correlations (bold lines) and key HMBC (H→C) correlations.
diastereotopic C12 protons to the carbonyl carbon of 3-
hydroxydodecanoic acid, confirming the structure of 1. A more
subtle difference in the NMR spectra of 1 is evident in the
distinct sets of chemical shifts observed for the three DHBA
units and four Ser residues, arising from the asymmetry of 1,
compared to the symmetry of enterobactin, which shows a
single set of resonances for the DHBA-Ser unit (Table S3,
Figures S10−S13).
The proposed amphi-enterobactin biosynthetic pathway
contains six genes (aebA−F) predicted to encode proteins
that are homologous to the well-characterized enterobactin
biosynthetic machinery (Figure 1, Table S1). In E. coli, EntC,
EntB (N-terminal domain), and EntA convert chorismate to
Figure 2. RP-HPLC chromatogram of amphi-enterobactin extraction
samples: wt BAA-1116 strain (red), ΔaebG (blue), ΔaebF (black).
Flow rate = 7 mL/min, solvent A = H2O, solvent B = MeOH.
2,3-dihydroxybenzoic acid (DHBA); EntE, EntB (C-terminal
domain), EntD, and EntF, four nonribosomal peptide
synthetase (NRPS) proteins, catalyze formation of the
fatty acids15,16 (Figure S4B). The ESI-MS/MS pattern is
DHBA-L-Ser unit, polymerize three of these units, and cyclize
the trimer to form the lactone backbone.8 However, a
distinguishing feature in V. harveyi is the presence of the
proximal aebG gene, predicted to encode a FACL. FACL
enzymes have been demonstrated to activate fatty acids to fatty
acyl-CoA thioesters prior to incorporation into acylated
nonribosomal peptides.9,10 Therefore, we predict that the
aebA−F gene cluster is responsible for the formation of the
amphi-enterobactin core structure and the aebG gene is
engaged in the acylation of the serine residue.
To investigate the postulated amphi-enterobactin biosyn-
thetic pathway, knockout mutants of the putative FACL (aebG)
and NRPS (aebF) genes were constructed in V. harveyi BAA-
1116, and the knockout strains were grown under iron-limiting
conditions to promote amphi-enterobactin production. RP-
HPLC analysis of the ΔaebG V. harveyi and ΔaebF V. harveyi
cell pellet and supernatant extracts reveals the abolition of the
amphi-enterobactins (Figure 2). Moreover, enterobactin was
not detected. AebG gene complementation restored amphi-
enterobactin production (Figure S15). The complementation
confirms that this gene is necessary for the production of the
acylated enterobactin siderophores. We propose that AebG is
required to activate the fatty acid as a fatty acid CoA thioester
consistent with the predicted amphi-enterobactin structure,
with fragmentation along the lactone backbone producing ions
containing both L-Ser-DHBA and L-Ser-fatty acid. The attached
fatty acid varied among the amphi-enterobactins in terms of
chain length (C10−C14), degree of unsaturation, and whether
the fatty acid was hydroxylated (Table S2).
The structure of amphi-enterobactin C12-OH (1; peak 4 in
Figure 2) was further elucidated with high-resolution electro-
1
spray ionization mass spectrometry (HIRESIMS) and H, 13C,
and 2D (COSY, TOCSY, HMBC) NMR (Figure 1D).
HIRESIMS established the mass of the molecular ion [M
+Na]+ of 1 as 977.3272 Da, corresponding to a molecular
formula of C45H54N4O19Na (Figure S9). NMR of 1 revealed
1
the H resonances to be similar to reported chemical shifts of
enterobactin,17 with the exception of the aliphatic peaks in the
1H spectrum of 1, resulting from the presence of the fatty acid.
Integration from 0.89 to 1.5 ppm corresponded to the 19
aliphatic protons on C14−C22. The proton of the hydroxy-
bearing carbon at C13 generated a multiplet at 3.91 ppm, and
the diastereotopic protons of C12 were split into a doublet of
multiplets at 2.27 and 2.34 ppm, authenticating the presence of
3-hydroxydodecanoic acid (Figure S10). HMBC correlations
showed the connectivity of both the α-1H serine and the
5616
dx.doi.org/10.1021/ja5019942 | J. Am. Chem. Soc. 2014, 136, 5615−5618

Journal of the American Chemical Society
Communication
Figure 3. Proposed biosynthesis of the amphi-enterobactin siderophores based on the iterative enterobactin “waiting room” model:18 C,
condensation domain; A, adenylation domain; T, thiolation domain; TE, thioesterase domain.
prior to incorporation9,10 and that AebF catalyzes amide bond
formation and cyclization of the lactone backbone.
to the well-characterized components of the enterobactin
synthetase, EntE and EntB (64% and 66% similarity,
respectively), AebE and AebB were predicted to be necessary
for DHBA activation, AebE functioning as the DHBA-AMP
ligase and AebB as the DHBA carrier protein. We hypothesized
that, once DHBA is loaded on the P-pant arm of AebB, the C
domain of AebF would catalyze DHBA amide formation with L-
Ser-S-P-pant-AebF, homologous to the C domain of the EntF
enterobactin synthetase.24 The aebE and aebB genes were
cloned and recombinantly expressed for in vitro studies. AebB
was obtained as a C-terminal His-tagged fusion protein and
AebE as a N-terminal thioredoxin-His tagged construct
(Figures S18 and S19). DHBA specific activation of AebE
was established through the nonradioactive malachite green
colorimetric assay.21,22 To determine the DHBA-Ser amidation
activity of AebF, holo-AebB, holo-AebF, and AebE were
incubated with L-Ser, DHBA, Mg2+, and ATP at room
temperature. However, following workup, no DHBA-Ser
product was detected. To determine if the fatty acid CoA
thioester is necessary to initiate the biosynthesis of the amphi-
enterobactin siderophores, lauroyl CoA was added to the
reaction mixture above and extracted with ethyl acetate after
incubation. The [M+H]+ amphi-enterobactin C12 ion was
detected at m/z 939.3 via LC-MS and LC-MS/MS, identifiable
by the fragmentation pattern of the tandem mass spectrum
(Figure S20). Control reactions without ATP did not produce
detectable amounts of siderophore.
In sum, we have isolated and characterized a suite of
unprecedented amphi-enterobactin siderophores, originally
predicted from the bioinformatic analysis of the V. harveyi
ATCC BAA-1116 genome. Eight amphi-enterobactins were
identified with various fatty acid appendages ranging in length
(C10−C14), degree of unsaturation, and hydroxylation,
dominated by amphi-enterobactin C12-OH, 1 (Figure 1D).
Amphi-enterobactin production is induced under low-iron
conditions, suggesting they are important for iron acquisition in
V. harveyi, and we propose that amphi-enterobactin production
is conserved among many marine Vibro spp. based on genome
Bioinformatic analysis of AebF, including sequence alignment
and structure prediction, indicates a linear four-domain NRPS
organization: C-A-T-TE (C, condensation; A, adenylation; T,
thiolation; TE, thioesterase), similar to the enterobactin NRPS,
EntF (54% similarity) (Table S1). Additionally, the A domain
of AebF contains the identical specificity-conferring residues as
EntF (DVWHFSLV), allowing prediction of L-Ser specific
activation.19,20 In the biosynthesis of enterobactin, the one C
domain of EntF catalyzes amide bond formation between
DHBA tethered to the 4′-phosphopantetheine (DHBA-S-P-
pant-EntB) arm of the aryl carrier protein, EntB, and L-Ser-S-P-
pant-EntF. However, in the amphi-enterobactins, two types of
amide bonds are present, L-Ser-DHBA and L-Ser-fatty acid,
while domain prediction analysis shows only one C domain. To
probe the activity of the ∼148 kDa AebF protein in vitro, the
aebF gene was cloned and recombinantly expressed in E. coli
with a C-terminal His-tag for purification (Figure S16). L-Ser
activation of AebF was tested using the nonradioactive
malachite green colorimetric assay,21,22 confirming the
predicted specificity. To evaluate the N-acylation activity of
the C domain, holo-AebF was generated in situ via treatment
with coenzyme A and recombinant P-pant transferase, Sfp,
from Bacillus subtilis.23 Condensation between lauroyl CoA and
L-serine was tested by incubating them with holo-AebF in the
presence of Mg2+ and ATP for 2 h at room temperature. After
hydrolysis of the acyl-serine from AebF with KOH, the product
was analyzed with LC-MS and LC-MS/MS. The [M+H]+
lauroyl-Ser ion was detected at m/z 288.2 (Figure S17). The
fragmentation pattern of the tandem spectrum is consistent
with lauroyl-Ser (Figure S17). The control reaction without
AebF produced no acylated product, demonstrating that the
sole NRPS C domain catalyzes the fatty acid acylation of L-
serine using fatty acyl-CoA thioester as a donor.
With AebF shown to be capable of producing the acylated
serine residue, DHBA-Ser amidation by the C domain of AebF
was examined. On the basis of the homology of AebE and AebB
5617
dx.doi.org/10.1021/ja5019942 | J. Am. Chem. Soc. 2014, 136, 5615−5618

Journal of the American Chemical Society
Communication
mining. An emerging characteristic of marine bacterial
siderophores is the prevalence of acyl siderophores comprised
of a peptidic iron-binding headgroup appended by one of a
series of long-chain fatty acids of varying degrees of saturation
and hydroxylation.5,15,16 Acyl siderophore precursors have also
recently been discovered in the biosynthesis of pyoverdine from
the human pathogen Pseudomonas aeruginosa.25,26 The fatty
acid group is proposed to anchor the acyl pyoverdine precursor
in the bacterial membrane, where the biosynthesis is suggested
to occur.26,27 Enterobactin biosynthetic proteins have also been
proposed to be membrane associated.28 While amphi-entero-
bactin biosynthetic proteins are predicted to be cytoplasmic,
the cellular location has not been experimentally determined,
and a membrane-associated biosynthetic strategy could take
place in V. harveyi similar to that in P. aeruginosa.
AUTHOR INFORMATION
Corresponding Author
butler@chem.ucsb.edu
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding from NSF CHE1059067 (A.B.) and OHSU Senior
Vice President for Research (M.G.H.) is gratefully acknowl-
edged. We thank H. Zhou (NMR) and J. Pavlovich (MS).
REFERENCES
■
(1) Lin, B.; Wang, Z.; Malanoski, A. P.; O’Grady, E. A.; Wimpee, C.
F.; Vuddhakul, V.; Alves, N., Jr.; Thompson, F. L.; Gomez-Gil, B.;
Vora, G. J. Environ. Microbiol. Rep. 2010, 2, 81.
The amphi-enterobactin biosynthetic enzymes are similar to
the enterobactin biosynthetic enzymes; however, we have
shown that the activity of the C domain on the NRPS protein,
AebF, differs. In the enterobactin synthetase, EntF, the C
domain catalyzes amide bond formation between DHBA-S-P-
pant-EntB and L-Ser-S-P-pant-EntF. Our analysis shows that
the C domain of the analogous amphi-enterobactin biosyn-
thetic protein, AebF, accepts both DHBA-S-P-pant-AebB and
fatty acyl-CoA thioesters as donors in the DHBA amidation and
fatty acid acylation of L-Ser-S-P-pant-AebF, demonstrating
novel bifunctional activity. We propose that the fatty acyl-
CoA thioester acylation of L-Ser-S-P-pant-AebF initiates
biosynthesis, based on the absence of DHBA-Ser product
formation when the fatty acid CoA thioester is omitted. We
also propose that AebG, a putative FACL located near the
cluster, provides the acyl-CoA thioester substrate. AebG is
necessary for biosynthesis, as demonstrated by the absence of
the amphi-enterobactin siderophores, as well as any DHBA-Ser
fragments, in the ΔaebG V. harveyi BAA-1116 cell pellet extract
or supernatant. The reaction catalyzed by AebF provides the
first biochemical demonstration of a C domain that performs
condensation reactions using two very different donor
substrates, although similar activity is predicted in the initial
C domain in the biosynthetic pathway of the acylated peptide
ramoplanin.29 Many acylated peptides have antimicrobial
properties that are highly dependent upon the nature of the
fatty acid appendage.30 Continued investigations into these
promiscuous starter C domains will provide a platform for
bioengineering new acylated peptides.
(2) Cano-Gomez, A.; Høj, L.; Owens, L.; Andreakis, N. Syst. Appl.
Microbiol. 2011, 34, 561.
(3) Lilley, B. N.; Bassler, B. L. Mol. Microbiol. 2000, 36, 940.
(4) Ng, W.-L.; Bassler, B. L. Annu. Rev. Genet. 2009, 43, 197.
(5) Sandy, M.; Butler, A. Chem. Rev. 2009, 109, 4580.
(6) Naka, H.; Actis, L. A.; Crosa, J. H. MicrobiologyOpen 2013, 2, 182.
(7) Weinberg, E. D. Biochim. Biophys. Acta 2009, 1790, 600.
(8) Crosa, J. H.; Walsh, C. T. Microbiol. Mol. Biol. Rev. 2002, 66, 223.
(9) Kraas, F. I.; Helmetag, V.; Wittmann, M.; Strieker, M.; Marahiel,
M. A. Chem. Biol. 2010, 17, 872.
(10) Imker, H. J.; Krahn, D.; Clerc, J.; Kaiser, M.; Walsh, C. T. Chem.
Biol. 2010, 17, 1077.
(11) Rosconi, F.; Davyt, D.; Martínez, V.; Martínez, M.; Abin-
Carriquiry, J. A.; Zane, H.; Butler, A.; de Souza, E. M.; Fabiano, E.
Environ. Microbiol. 2013, 15, 916.
(12) Kreutzer, M. F.; Kage, H.; Nett, M. J. Am. Chem. Soc. 2012, 134,
5415.
(13) Schwyn, B.; Neilands, J. B. Anal. Biochem. 1987, 160, 47.
(14) Berner, I.; Greiner, M.; Metzger, J.; Jung, G.; Winkelmann, G.
Biol. Metals 1991, 4, 113.
(15) Martinez, J. S.; Zhang, G. P.; Holt, P. D.; Jung, H. T.; Carrano,
C. J.; Haygood, M. G.; Butler, A. Science 2000, 287, 1245.
(16) Martinez, J. S.; Carter-Franklin, J. N.; Mann, E. L.; Martin, J. D.;
Haygood, M. G.; Butler, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
3754.
(17) Fiedler, H.-P.; Krastel, P.; Muller, J.; Gebhardt, K.; Zeeck, A.
̈
FEMS Microbiol. Lett. 2001, 196, 147.
(18) Shaw-Reid, C. A.; Kelleher, N. L.; Losey, H. C.; Gehring, A. M.;
Berg, C.; Walsh, C. T. Chem. Biol. 1999, 6, 385.
(19) Stachelhaus, T.; Mootz, H. D.; Marahiel, M. A. Chem. Biol.
1999, 6, 493.
(20) Challis, G. L.; Ravel, J.; Townsend, C. A. Chem. Biol. 2000, 7,
On the basis of the results of this study, we propose a
putative amphi-enterobactin biosynthetic pathway constructed
off the enterobactin “waiting room” model of biosynthesis18
(Figure 3). In this model, the TE domain serves as a “waiting
room” for the L-Ser-DHBA during each iterative round. For the
biosynthesis of the amphi-enterobactins, the TE domain of
AebF must be able to harbor the 16-member tetralactone
backbone of the amphi-enterobactins, versus the 12-member
trilactone of enterobactin, suggesting an increased degree of
elasticity for the TE domain of AebF. Future crystallographic
data will allow for the structural comparison of TE domains and
may provide insight into the unique characteristics of AebF.
211.
(21) McQuade, T. J.; Shallop, A. D.; Sheoran, A.; DelProposto, J. E.;
Tsodikov, O. V.; Garneau-Tsodikova, S. Anal. Biochem. 2009, 386, 244.
(22) Xia, S.; Ma, Y.; Zhang, W.; Yang, Y.; Wu, S.; Zhu, M.; Deng, L.;
Li, B.; Liu, Z.; Qi, C. PLoS One 2012, 7, e37487.
(23) Quadri, L. E. N.; Weinreb, P. H.; Lei, M.; Nakano, M. M.;
Zuber, P.; Walsh, C. T. Biochemistry 1998, 37, 1585.
(24) Gehring, A. M.; Mori, I.; Walsh, C. T. Biochemistry 1998, 37,
2648.
(25) Drake, E. J.; Gulick, A. M. ACS Chem. Biol. 2011, 6, 1277.
(26) Hannauer, M.; Schafer, M.; Hoegy, F.; Gizzi, P.; Wehrung, P.;
Mislin, G. L. A.; Budzikiewicz, H.; Schalk, I. J. FEBS Lett. 2012, 586,
96.
(27) Imperi, F.; Visca, P. FEBS Lett. 2013, 587, 3387.
(28) Hantash, F. M.; Earhart, C. F. J. Bacteriol. 2000, 182, 1768.
(29) Hoertz, A. J.; Hamburger, J. B.; Gooden, D. M.; Bednar, M. M.;
McCafferty, D. G. Bioorg. Med. Chem. 2012, 20, 859.
(30) Baltz, R. H.; Miao, V.; Wrigley, S. K. Nat. Prod. Rep. 2005, 22,
717.
̈
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and characterization data for 1. This
material is available free of charge via the Internet at http://
pubs.acs.org.
5618
dx.doi.org/10.1021/ja5019942 | J. Am. Chem. Soc. 2014, 136, 5615−5618