Biosynthesis of Amphi-enterobactin Siderophores

Biosynthesis of Amphi-enterobactin Siderophores by Vibrio harveyi BAA-1116:Identi?cation 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,California93106-9510,United States

?Institute of Environmental Health,Oregon Health&Science University,Portland,Oregon97239-3098,United States

§Departamento de Bioquímica y Geno m ica Microbianas,Instituto de Investigaciones Biolo g icas Clemente Estable,Montevideo 11600,Uruguay

*Supporting Information

Vibrio harveyi BAA-1116(recently reclassi?ed as V.campbel-lii1,2)is widely used to study quorum sensing,due to its quorum-regulated bioluminescence.3,4Quorum 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.5Side-rophores contribute to competitiveness in environmental bacteria and serve as virulence factors in pathogens,including Vibrios.6Microbial colonization of an animal host depends critically on the ability to obtain su?cient iron,yet is also challenged by the low levels of available iron in the host environment.7

The genome of V.harveyi strain ATCC BAA-1116contains a nonribosomal peptide synthetase(NRPS)gene cluster resembling that for enterobactin biosynthesis(Figure1,Table S1),yet we did not detect enterobactin in cultures of V.harveyi.Enterobactin,the lactone of tris-2,3-dihydroxybenzoyl-L-serine, is the prototypic catecholate siderophore produced by many bacteria,most notably Escherichia coli8(Figure1C).An

encoded long-chain fatty acid CoA ligase(FACL)is present

in the V.harveyi genome near the putative enterobactin biosynthetic gene cluster(Figure1B).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,speci?cally

the FACL nearby the NRPS genes,in conjunction with the continued discovery of new N-terminally acylated peptide

siderophores,11,12led 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 (Figure1B,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-1116was grown in a low-iron

synthetic seawater medium.After2days,the culture was harvested by centrifugation,the bacterial cell pellet was extracted with methanol,and the extract was puri?ed with solid-phase adsorption and RP-HPLC(λ=310nm for catechol absorption).Eight peaks gave a positive CAS assay response for iron binding,consistent with the presence of apo-siderophores (Figure2).13ESI-MS of these compounds showed a range in mass from m/z927to m/z987(Table S2,Figures S1?S8). Enterobactin(m/z670[M+H]+)was not detected in either the culture supernatant or the bacterial cell pellet.

ESI-MS/MS of the eight compounds produced a similar

pattern,with three major daughter ions at m/z670,m/z447, and m/z224([M+H]+)(Figure S4A),as observed in the ESI-MS/MS of enterobactin.14However,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 di?ering

Received:February26,2014

Published:April4,2014

fatty acids 15,16(Figure S4B).The ESI-MS/MS pattern is 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 4in Figure 2)was further elucidated with high-resolution electro-spray ionization mass spectrometry (HIRESIMS)and 1H,13C,and 2D (COSY,TOCSY,HMBC)NMR (Figure 1D).HIRESIMS established the mass of the molecular ion [M +Na]+of 1as 977.3272Da,corresponding to a molecular formula of C 45H 54N 4O 19Na (Figure S9).NMR of 1revealed the 1H 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 0.89to 1.5ppm corresponded to the 19aliphatic protons on C14?C22.The proton of the hydroxy-bearing carbon at C13generated a multiplet at 3.91ppm,and the diastereotopic protons of C12were split into a doublet of multiplets at 2.27and 2.34ppm,authenticating the presence of 3-hydroxydodecanoic acid (Figure S10).HMBC correlations showed the connectivity of both the α-1H serine and the diastereotopic C12protons to the carbonyl carbon of 3-hydroxydodecanoic acid,con ?rming the structure of 1.A more subtle di ?erence in the NMR spectra of 1is 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 2,3-dihydroxybenzoic acid (DHBA);EntE,EntB (C-terminal domain),EntD,and EntF,four nonribosomal peptide synthetase (NRPS)proteins,catalyze formation of the DHBA-L -Ser unit,polymerize three of these units,and cyclize the trimer to form the lactone backbone.8However,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.9,10Therefore,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 con ?rms 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

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 S1for details).Boxes with dashed lines indicate the genetic regions deleted in this study.(C)Enterobactin.(D)Amphi-enterobactin C12-OH (1;peak 4in Figure 2)depicted with 1H ?1H correlations (bold lines)and key HMBC (H →C)

correlations.

Figure 2.RP-HPLC chromatogram of amphi-enterobactin extraction samples:wt BAA-1116strain (red),ΔaebG (blue),ΔaebF (black).Flow rate =7mL/min,solvent A =H 2O,solvent B =MeOH.

prior to incorporation 9,10and that AebF catalyzes amide bond formation and cyclization of the lactone backbone.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 speci ?city-conferring residues as EntF (DVWHFSLV),allowing prediction of L -Ser speci ?c activation.19,20In 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 ～148kDa AebF protein in vitro ,the aebF gene was cloned and recombinantly expressed in E.coli with a C-terminal His-tag for puri ?cation (Figure S16).L -Ser activation of AebF was tested using the nonradioactive malachite green colorimetric assay,21,22con ?rming the predicted speci ?city.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 .23Condensation between lauroyl CoA and L -serine was tested by incubating them with holo -AebF in the presence of Mg 2+and ATP for 2h 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 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.24The 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 S18and S19).DHBA speci ?c activation of AebE was established through the nonradioactive malachite green colorimetric assay.21,22To determine the DHBA-Ser amidation activity of AebF,holo -AebB,holo -AebF,and AebE were incubated with L -Ser,DHBA,Mg 2+,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 C12ion was detected at m /z 939.3via LC-MS and LC-MS/MS,identi ?able 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-1116genome.Eight amphi-enterobactins were identi ?ed 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

Figure 3.Proposed biosynthesis of the amphi-enterobactin siderophores based on the iterative enterobactin “waiting room ”model:18C,condensation domain;A,adenylation domain;T,thiolation domain;TE,thioesterase domain.

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.5,15,16Acyl siderophore precursors have also recently been discovered in the biosynthesis of pyoverdine from the human pathogen Pseudomonas aeruginosa .25,26The fatty acid group is proposed to anchor the acyl pyoverdine precursor in the bacterial membrane,where the biosynthesis is suggested to occur.26,27Enterobactin biosynthetic proteins have also been proposed to be membrane associated.28While 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 .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,di ?ers.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-1116cell pellet extract or supernatant.The reaction catalyzed by AebF provides the ?rst biochemical demonstration of a C domain that performs condensation reactions using two very di ?erent donor substrates,although similar activity is predicted in the initial C domain in the biosynthetic pathway of the acylated peptide ramoplanin.29Many acylated peptides have antimicrobial properties that are highly dependent upon the nature of the fatty acid appendage.30Continued investigations into these promiscuous starter C domains will provide a platform for bioengineering new acylated peptides.On the basis of the results of this study,we propose a putative amphi-enterobactin biosynthetic pathway constructed o ?the enterobactin “waiting room ”model of biosynthesis 18(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.■ASSOCIATED CONTENT *Supporting Information Experimental details and characterization data for 1.This material is available free of charge via the Internet at https://www.sodocs.net/doc/d316963926.html,.

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