Advertisement

JBIC Journal of Biological Inorganic Chemistry

, Volume 23, Issue 7, pp 1009–1022 | Cite as

Amphi-enterobactin commonly produced among Vibrio campbellii and Vibrio harveyi strains can be taken up by a novel outer membrane protein FapA that also can transport canonical Fe(III)-enterobactin

  • Hiroaki Naka
  • Zachary L. Reitz
  • Aneta L. Jelowicki
  • Alison Butler
  • Margo G. Haygood
Original Paper
Part of the following topical collections:
  1. Alison Butler: Papers in Celebration of Her 2018 ACS Alfred Bader Award in Bioorganic or Bioinorganic Chemistry

Abstract

Vibrio campbellii BAA-1116 (formerly Vibrio harveyi) is a model organism for quorum sensing study and produces the siderophores anguibactin and amphi-enterobactin. This study examined the mechanisms and specificity of siderophore uptake in V. campbellii and V. harveyi, and surveyed the diversity of siderophore production in V. campbellii and V. harveyi strains. The amphi-enterobactin gene cluster of BAA-1116 harbors a gene, named fapA, that is a homologue of genes encoding Fe(III)-siderophore-specific outer membrane receptors. Another strain, V. campbellii HY01, a strain pathogenic to shrimp, also carries this cluster including fapA. Our siderophore bioassay results using HY01-derived indicator strains show that the FapA protein localized in the outer membrane fraction of V. campbellii HY01 is essential for the uptake of Fe(III)-amphi-enterobactin as well as exogenous siderophores, including enterobactin from E. coli, but not vanchrobactin from V. anguillarum RV22 while Fe(III)-amphi-enterobactin can be utilized by V. anguillarum. Electrospray ionization mass spectrometry as well as bioassay revealed that various V. campbellii and V. harveyi strains produce a suite of amphi-enterobactins with various fatty acid appendages, including several novel amphi-enterobactins, and these amphi-enterobactins can be taken up by V. campbellii HY01 via FapA, indicating that amphi-enterobactin production is a common phenotype among V. campbellii and V. harveyi, whereas our previous work, confirmed herein, showed that anguibactin is only produced by V. campbellii strains. These results along with the additional finding that a 2,3-dihydroxybenzoic acid biosynthesis gene, aebA, located in the amphi-enterobactin gene cluster, is essential for both anguibactin and amphi-enterobactin biosynthesis, suggest the possibility that amphi-enterobactin is a native siderophore of V. campbellii and V. harveyi, while the anguibactin system has been acquired by V. campbellii during evolution.

Keywords

Vibrio campbellii Vibrio harveyi Amphi-enterobactin Anguibactin Fe(III)-siderophore receptor 

Introduction

Iron is an indispensable metal for almost all living organisms including bacteria because it is involved in many biological processes. Although iron is the fourth most abundant metal in the Earth’s crust, iron is not readily available since it is nearly insoluble at neutral pH in the presence of oxygen, and it is strongly bound to ligands in environmental and host conditions [1]. Thus, bacteria require high affinity transport systems to overcome these iron limiting conditions to survive. One of the strategies bacteria have evolved is the siderophore-mediated iron transport system in which bacteria produce siderophores, small molecule iron chelators, and take up Fe(III)-siderophores via cognate outer membrane specific receptors [2, 3].

The marine bacterium Vibrio harveyi, a part of the Harveyi clade that is a main group within the genus Vibrio, is ubiquitous in seawater; some of these strains are pathogens to marine vertebrates and invertebrates [4, 5]. V. campbellii is another Harveyi clade bacterium commonly known to be a pathogen of marine organisms, especially shrimp [6, 7, 8, 9]. Since V. campbellii and V. harveyi are phenotypically closely related and their ribosomal RNA gene sequences are very similar, discriminating between these two species has been challenging. Lin et al. [10] used microarray-based comparative genomic hybridization (CGH) and multilocus sequence analyses (MLSA) to show that many strains thought to be V. harveyi, including BAA-1116, a model organism for quorum sensing [11, 12], actually belong to V. campbellii. The availability of large numbers of genome sequences of bacteria due to recent advances in sequencing technology provided further details of phylogenetic relationships among the Harveyi clade [13, 14, 15].

We previously showed that V. campbellii BAA-1116 and HY01 (formerly V. harveyi [10, 15]) produce two siderophores, anguibactin and amphi-enterobactin [16, 17] (Fig. 1). Anguibactin is a siderophore originally found as an important virulence factor in the fish pathogen V. anguillarum, and the anguibactin biosynthesis and transport gene cluster is located in the 65-kb virulence plasmid pJM1 [18, 19]. We previously reported that anguibactin is commonly produced by various V. campbellii strains but not V. harveyi strains, and the anguibactin biosynthesis and transport gene cluster including a nonribosomal peptide synthetase gene angR that are essential for anguibactin biosynthesis, and the fatA gene encoding the Fe(III)-anguibactin specific outer membrane receptor, are located on the chromosome of V. campbellii BAA-1116 and HY01 (Fig. 2) [17]. The other siderophore, amphi-enterobactin, is a siderophore that has a fatty acid attached to an enterobactin-like molecule (Fig. 1) [16]. We showed that the aebF gene encoding a nonribosomal peptide synthetase and the aebG gene encoding a long chain fatty acid CoA ligase is essential for amphi-enterobactin biosynthesis. V. campbellii DS40M4 produces anguibactin, as well as the monomer, dimer and trimer of vanchrobactin (Fig. 1) [20]. Vanchrobactin was originally found in the V. anguillarum pJM1-less strain RV22 [21, 22], and genome sequence data of the DS40M4 strain indicate that a potential vanchrobactin cluster that is similar to that of RV22 might be responsible for trivanchrobactin biosynthesis [23]. Recently, Thode et al. [24] reported that homologues of the amphi-enterobactin biosynthetic genes can be found in genome sequences of V. campbellii and V. harveyi. However, their analysis does not provide information whether the amphi-enterobactin biosynthetic genes are found in some specific strains or widespread among V. campbellii and V. harveyi strains, and the amphi-enterobactin transport system has not been identified yet. Table 1 shows the presence of anguibactin biosynthesis (angR) and transport (fatA), and amphi-enterobactin biosynthesis (aebF and aebG) and transport (fapA) genes among genome sequences of V. campbellii and V. harveyi used in this study.
Fig. 1

The structure of siderophores discussed in this study

Fig. 2

The anguibactin and amphi-enterobactin gene clusters in V. campbellii HY01. Genes involved in siderophore biosynthesis and transport were shown by blue-filled arrows and orange-filled arrows, respectively, and the dotted lines indicate that the genes aebA, C, E and B are involved in the biosynthesis of 2,3-dihydroxybenzoic acid (DHBA) that is a precursor of both anguibactin and amphi-enterobactin

Table 1

Distribution of proven anguibactin and amphi-enterobactin biosynthesis and transport genes in V. harveyi and V. campbellii strains used in this study

Strain

species

Former species

Anguibactin genes

Amphi-enterobactin genes

angR

fatA

aebF

aebG

fapA

HY01

V. campbellii

V. harveyi

+

+

+

+

+

BAA-1116

V. campbellii

V. harveyi

+

+

+

+

+

42A

V. campbellii

NA

+

+

+

+

+

CAIM 115

V. campbellii

NA

+

+

+

+

+

CAIM 198

V. campbellii

NA

+

+

+

+

+

CAIM 519T

V. campbellii

NA

+

+

+

+

+

DS40M4

V. campbellii

Vibrio sp.

+

+

+

+

+

CAIM 148

V. harveyi

NA

+

+

+

CAIM 513T

V. harveyi

NA

+

+

+

CAIM 1075

V. harveyi

NA

+

+

+

CAIM 1792

V. harveyi

NA

+

+

+

NA no change

+, presence; −, absence. The genes angR and fatA are essential for anguibactin biosynthesis and ferric-anguibactin transport, respectively. The genes aebF and aebG are essential for amphi-enterobactin transport while the fapA gene is essential for ferric-amphi-enterobactin uptake

Our long-term goal is to understand how marine bacteria have evolved siderophore-mediated iron transport systems in aquatic environments. In this study, we used two closely related bacteria, V. campbellii and V. harveyi, as models to understand mechanisms, distribution and evolution of siderophore-mediated iron transport mechanisms, and provide an example of how marine bacteria use siderophore systems to compete for iron using V. campbellii and another marine bacteria V. anguillarum.

Materials and methods

Strains and growth conditions

Strains and plasmids used in this study are listed in Table S1. E. coli strains were grown in LB medium. V. campbellii, V. harveyi and V. anguillarum strains were grown in Luria Marine (LM) medium containing LB broth (Difco, Sparks, MD) and 1.5% NaCl. AB medium [25] was used for growth experiments and siderophore bioassays. Agar (1.5%) and Agar Noble (0.75 or 1%) were added into LB and LM, and AB medium, respectively, to prepare agar plates. Antibiotics were added in the medium if needed, with following conditions; ampicillin at 100 µg/mL for E. coli, chloramphenicol at 30 µg/mL for E. coli and at 10 µg/mL for V. campbellii. Iron chelators such as 2,2′-dipyridyl and ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA) were supplemented in the growth medium to generate iron limiting conditions. EDDA was purchased from Complete Green Company (EL Segundo, CA) and iron was removed as previously described [26]. Since 2,2′-dipyridyl is more stable as an iron(III) chelator than EDDA, 2,2′-dipyridyl was normally used to culture V. campbellii in liquid medium; EDDA was used in the bioassay because it provided stable results with positive and negative controls in a much wider range of concentrations as compared with 2,2′-dipyridyl.

Detection of amphi-enterobactin by electrospray ionization mass spectrometry (ESI–MS)

Strains were grown in a low-iron artificial seawater medium containing casamino acids (10 g/L), NH4Cl (19 mM), disodium hydrogen phosphate (4.6 mM), MgSO4 (50 mM), CaCl2(10 mM), trace metal grade NaCl (0.3 M), KCl (10 mM), glycerol (41 mM), HEPES buffer (10 mM; pH 7.4), NaHCO3 (2 mM), biotin (8.2 μM), niacin (1.6 μM), thiamin (0.33 μM), 4-aminobenzoic acid (1.46 μM), pantothenic acid (0.21 μM), pyridoxine hydrochloride (5 μM), cyanocobalamin (0.07 μM), riboflavin (0.5 μM), and folic acid (0.5 μM) [16]. Cultures were grown at 100 mL scale in 250-mL acid-washed erlenmeyer flasks on an orbital shaker (180 rpm). After 48 h, cultures were harvested by centrifugation (5400 RCF, 15 min). The cell pellet was resuspended in ethanol (30 mL per pellet) and shaken overnight at 4 °C. The ethanol extract was centrifuged briefly (13,000 rpm, 5 min) and filtered through a 0.22-µm membrane. Extracts were analyzed through positive ion mode ESI–MS on a Waters Xevo G2-XS QTof coupled to a Waters Acquity H-Class UPLC system. A Waters BEH C18 column was used with a gradient of 50–90 or 100% acetonitrile/water (both with 0.1% w/v formic acid). Using MassLynx 4.1, chromatograms for masses of interest were generated and molecular ion peaks quantified by integration (ApexTrack algorithm).

Construction of deletion mutants and complementation

Primers used in this study are listed in Table S2. To construct ∆aebF, splicing by overhang extension (SOE)-PCR was used to combine flanking regions of the target genes to be mutated [27]. The deletion fragments thus obtained were ligated into pGEM-T easy (Promega), transformed into E. coli DH5α and DNA sequences of the inserted DNA were confirmed by sequencing. To construct ∆fapA and ∆aebA, upstream and downstream regions were PCR-amplified and cloned into pGEM-T easy (Promega). After confirmation of DNA sequence of the inserted DNA, the upstream and downstream regions were cloned together in pBluescript II. Plasmids containing the deletion fragments were restriction enzyme-digested and the deletion fragments were cloned into suicide vector pDM4 digested with the corresponding restriction enzymes. The pDM4-derivatives were conjugated from E. coli S17-1λpir into V. campbellii by biparental mating as described previously [17]. First recombinants were selected by plating exconjugants on TCBS plates containing 10 µg/mL chloramphenicol. First recombinants thus obtained were grown in LM broth without antibiotics and subsequently streaked on LM plates supplemented with 15% sucrose to obtain second recombinants. Deletion mutants were selected by screening second recombinants by colony PCR. To complement deletion mutants, wild-type genes including putative ribosomal binding sites were cloned into pMMB208, and the pMMB208 derivatives were conjugated from E. coli S17-1λpir to V. campbellii. Construction of the ∆fvtA and ∆fvtA single mutants in V. anguillarum H775-3 and complementation of deletion mutants were followed by the procedures we previously described [28].

Siderophore bioassay

Siderophore bioassays (cross-feeding assays) were performed to test Fe(III)-siderophore utilization as described before with some modifications [17]. 2 × AB broth and 1.5% (or 2%) Agar Noble (Difco) was separately prepared and autoclaved. Equal volumes of these solutions were subsequently mixed and supplemented with 1 mM l-arginine, 1% glycerol and 10 mM potassium phosphate buffer (pH 7.0). The temperature of the medium was adjusted to approximately 40 °C, and the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA) was added to generate iron limiting conditions. Indicator strains (5 μL/mL) grown in LM broth overnight at room temperature were added into the medium, and gently mixed. If needed, chloramphenicol (10 µg/mL) and IPTG (1 mM) were also supplemented in the medium. After solidification, 5 µL of siderophore-producing bacteria grown overnight in LM broth at room temperature (or 1 µL of ferric ammonium citrate as a positive control) were spotted on the plates. After an appropriate period of incubation at ambient temperature (approximately 23 °C), growth halos around the spots were monitored.

Outer membrane extraction and SDS-PAGE

Vibrio campbellii HY01 derivatives were grown in 30 mL AB medium supplemented with 10 µg/mL chloramphenicol, 1 mM IPTG and 30 µM iron chelator 2,2′-dipyridyl until exponential phase (OD600 approximately 0.2), and outer membrane proteins were extracted using the N-lauroylsarcosine (sarkosyl) method as described previously [28]. To use as a negative control, HY01∆angRaebFfatA(pMMB208) was also grown under iron-rich conditions generated by supplementing 10 µg/mL ferric ammonium citrate instead of 30 µM 2,2′-dipyridyl. Extracted outer membrane proteins were resuspended into ddH2O and mixed with XT Sample buffer (Bio-Rad) and 5% 2-mercaptoethanol, adjusting the total volume to 40 µL. After boiling for 10 min, 20 µL of samples were separated by SDS-PAGE using 4–15% Mini-Protean TGX Precast Gels (Bio-Rad) with XT MOPS buffer (Bio-rad) for 2 h at 80 V, and stained with Bio-Safe™ Comassie G-250 Stain (Bio-Rad). Broad-Range SDS-PAGE Standards (Bio-Rad) was used as a molecular marker.

Arnow’s assay

Vibrio campbellii HY01 derivatives were grown overnight at ambient temperature in AB broth supplemented with 10 µg/mL chloramphenicol, 1 mM IPTG and 30 µM DIP. After centrifugation, supernatants were subjected to Arnow’s assay as described previously [29], and the obtained data were normalized by the OD600 values.

Preparation of crude extracts from V. campbellii HY01 derivatives

Vibrio campbellii HY01 derivatives were grown overnight at ambient temperature in 50 ml AB broth supplemented with 10 µg/mL chloramphenicol and 1 mM IPTG. After centrifugation of overnight culture, supernatants and cell pellets were separated. The supernatants were incubated with Amberlite XAD7HP resin (approximately 1 g) with agitation overnight at 4 °C. Filtered resin was washed by milliQ water and extracts were eluted by 100% methanol. The cell pellets were extracted with 100% methanol with agitation overnight at 4 °C. The supernatant and cell pellet extracts were then combined, and evaporated under reduced pressure. The dried extracts were resuspended in 100% methanol (adjusted to 10 mg/mL) and used for the siderophore bioassay.

Results

Identification of a candidate for the amphi-enterobactin outer membrane receptor gene

Amphi-enterobactin biosynthesis genes were previously identified in V. campbellii (formerly V. harveyi) BAA-1116 [16]. The genes aebA, C, E, B, F and D are located in the amphi-enterobactin cluster while the aebG gene is located 15 kbp away from this cluster (Fig. 2). Comparative genomic analysis indicated that the amphi-enterobactin cluster is well-conserved in V. campbellii HY01. Since HY01 grows faster than BAA-1116, and HY01-derived mutants in anguibactin production and uptake genes are available, we used HY01 as a model strain to characterize the amphi-enterobactin system. To confirm the production of amphi-enterobactin by HY01, we analyzed extracts prepared from V. campbellii HY01. The results in Table 2 confirm that V. campbellii HY01 produces a suite of four amphi-enterobactins. Furthermore, we constructed the ∆aebF mutant and showed that it does not produce amphi-enterobactin. Blast analysis of surrounding genes of the amphi-enterobactin biosynthetic gene cluster of HY01 identified a gene (A1Q_RS16075; VIBHAR_RS06650 in BAA-1116) annotated as a colicin I receptor gene (named fapA, Fig. 2) located just downstream of the aebD gene to be a homologue of genes encoding TonB-dependent outer membrane receptors. The identity and similarity of FapA proteins between BAA-1116 and HY01 is 95 and 96%, respectively. Further BlastP analysis showed that HY01 FapA has similarity to Fe(III)-siderophore-specific outer membrane receptors such as the E. coli enterobactin FepA receptor (26% identity, 44% similarity), V. anguillarum FvtA (23% identity, 36% similarity) and FetA (31% identity, 52% similarity) receptors, and P. aeruginosa PAO1 pyoverdine FpvA (24% identity, 37% similarity) and pyochelin FptA (22% identity, 35% similarity) receptors. Protein structure prediction using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) also chose the Fe(III)-pyoverdine outer membrane receptor (FpvA) as a top model. These results indicate that the fapA gene has the potential to encode a Fe(III)-amphi-enterobactin outer membrane receptor.
Table 2

Relative abundance of amphi-enterobactins among Vibrio campbellii and harveyi strains

Strain

Fatty acid tail:

10:0 OH

12:1 OH

12:0 OH

10:0a

14:1 OH

12:1

14:0 OHa

12:0

14:1

14:0a

16:1a

Species

m/z 927

m/z 953

m/z 955

m/z 911

m/z 981

m/z 937

m/z 983

m/z 939

m/z 965

m/z 967

m/z 993

HY01

V. campbellii

+

+

+

+

HY01ΔaebF

V. campbellii

42A

V. campbellii

+++

++

+++

+

CAIM 115

V. campbellii

++

++

+

+

CAIM 198

V. campbellii

++

+

++++

++

++

+++

++

++++

++++

++++

+++

CAIM 519

V. campbellii

+++

++

+++++

+++

+++

++++

++

+++++

+++++

++++

++++

DS40M4

V. campbellii

+

++

+

+

++

+

++++

+++

+++

++

CAIM 148

V. harveyi

+

+++

++

++

++

CAIM 513

V. harveyi

+

+

+

*

CAIM 1075

V. harveyi

++

+

+

+

+++

++

++

++

CAIM 1792

V. harveyi

++

++

+

+

UPLC/ESI-MS molecular ion counts were integrated and normalized to an OD of 1.0. +++++, > 106 normalized counts; ++++, > 105 normalized counts; +++, > 104 normalized counts; ++, > 103 normalized counts;; +,>102 normalized counts; *, < 102 normalized counts; −, not detected above background

Integrations are shown in Figs. S2 and S3

aFatty acid tails newly reported herein

Siderophore bioassay

In addition to amphi-enterobactin, V. campbellii HY01 produces anguibactin [17]. To assess amphi-enterobactin transport, we constructed siderophore production minus strains. An amphi-enterobactin biosynthesis gene, aebF (Fig. 2), was deleted in the ∆angR strain that does not synthesize anguibactin, generating a siderophore production minus strain, HY01∆aebFangR. We then mutated the fapA gene in the ∆aebFangR double mutant to evaluate the involvement of fapA in Fe(III)-amphi-enterobactin transport. As shown in Fig. 3, V. campbellii HY01∆angRaebF was able to overcome iron-limiting conditions and grew by taking up Fe(III)-siderophores from V. campbellii HY01 (anguibactin and amphi-enterobactin), V. campbellii HY01∆aebF (anguibactin) and HY01∆angR (amphi-enterobactin). When the fapA gene was mutated, we did not observe the cross-feeding of the indicator strain with HY01∆angR (amphi-enterobactin) while the mutation did not affect cross-feeding with anguibactin producers such as HY01 and HY01∆aebF. Expression of the fapA gene in trans from pMMB208 in the fapA mutant recovered their growth with the amphi-enterobactin producer HY01∆angR. These results indicate that the fapA gene is essential for Fe(III)-amphi-enterobactin uptake and the Fe(III)-anguibactin receptor FatA cannot compensate for the fapA mutant; thus FapA is the only Fe(III)-amphi-enterobactin receptor in HY01. We also tested whether FapA can transport exogeneous siderophores such as enterobactin from E. coli DH5a and vanchrobactin from V. anguillarum RV22. As shown in Fig. 3, it is clear that HY01∆angRaebF could utilize Fe(III)-enterobactin but not Fe(III)-vanchrobactin as an iron source. The fapA deletion mutant was not able to use Fe(III)-enterobactin and the complemented strains showed the recovery of cross-feeding. These results demonstrate that FapA can transport canonical enterobactin but not vanchrobactin.
Fig. 3

The fapA gene is responsible for Fe(III)-amphi-enterobactin transport. Indicator strains such as V. campbellii HY01∆angRaebF(pMMB208), V. campbellii HY01∆angRaebFfapA(pMMB208) and V. campbellii HY01∆angRaebFfapA(pMMB208-fapA) were mixed with melted AB agar supplemented with 1 mM IPTG and 10 µg/mL chloramphenicol. 5 µL of siderophore producers such as anguibactin [Ang] and amphi-enterobactin [Amphi-ent] producer, V. campbellii HY01(pMMB208); amphi-enterobactin producer, V. campbellii HY01∆angR(pMMB208); anguibactin producer, V. campbellii HY01∆aebF(pMMB208); enterobactin [Ent] producer, E. coli DH5α (pMMB208); vanchrobactin [Van] producer, V. anguillarum RV22(pMMB208) were spotted on the plates and incubated at ambient temperature (approximately 23 °C). Appearance of growth halos around spots was monitored after 16 h incubation until 5 days, and the results were consistent during monitoring. The pictures were taken after 40 h incubation. The smaller halo around the spot of E. coli DH5α (pMMB208) was most likely due to much slower growth speed of this strain as compared with V. campbellii at this growth condition

Localization of FapA in the outer membrane fractions

The signal peptide cleavage site predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) is located between amino acids number 21 and 22 of FapA, with the remaining 69.01 kDa protein as the predicted mature FapA. To test whether FapA protein is actually located in the outer membrane of V. campbellii, outer membrane proteins extracted using Sarkosyl from V. campbellii strains were compared by SDS-PAGE. To avoid complications from anguibactin and/or amphi-enterobactin production affecting the iron content or availability in the growth medium and to remove Fe(III)-anguibactin outer membrane receptor FatA background in the outer membrane fractions, we constructed V. campbellii HY01∆angRaebFfatA by mutating the aebF gene in the anguibactin production and uptake minus strain, HY01∆angRfatA [17]. The fapA gene was further mutated in the triple mutant, generating HY01∆angRaebFfatAfapA. The SDS-PAGE results shown in Fig. S1 indicate that under iron-limiting growth conditions generated by adding the iron chelator 2,2′-dipyridyl, the parent strain harboring empty pMMB208 showed a protein band that corresponds to the predicted size of mature FapA (lane 1) while this band did not appear in the iron-rich growth condition (lane 4), indicating that FapA is up-regulated under iron limiting conditions. This band was not observed when the fapA gene was deleted (lane 2), and the expression of the fapA gene from pMMB208 in the ∆fapA mutant recovered the phenotype showing a very intense band due to overexpression of the fapA gene from the tac promoter (lane 3). From these results, it is clear that the iron-regulated FapA protein is localized in the outer membrane of V. campbellii as predicted for a siderophore outer membrane receptor.

Amphi-enterobactin production from various V. campbellii and V. harveyi strains

The amphi-enterobactin cluster was originally identified in BAA-1116 [16]. Via a bioinformatic approach, Thode et al. [24] reported that the amphi-enterobactin biosynthetic genes can be found in V. campbellii and V. harveyi. However, it was still unknown whether the amphi-enterobactin gene cluster is specifically found in some strains or widespread among these species. Genome sequences of many V. campbellii and V. harveyi strains are currently available due to recent advances in sequencing technology. Analysis of all available genome sequences (31 V. campbellii and 33 V. harveyi strains) in Genbank revealed that the presence of anguibactin genes only in V. campbellii strains is consistent, and the amphi-enterobactin cluster can be found in both V. campbellii and V. harveyi strains (data not shown). However, actual production of amphi-enterobactin from various strains of V. campbellii and V. harveyi has not been tested yet. Since V. campbellii strains classified based on genome-wide analysis also produce anguibactin [17], we used the Fe(III)-anguibactin receptor gene fatA minus strains constructed in the previous section, HY01∆angRaebFfatA and HY01∆angRaebFfatAfapA as indicator strains to detect only amphi-enterobactin production. We previously reported that when two siderophores, anguibactin and vanchrobactin, are produced in V. anguillarum, the presence of anguibactin negatively affects the detection of vanchrobactin by cross-feeding assay due to chelation of iron from vanchrobactin by anguibactin [30]. Thus, we decided first to evaluate whether these indicator strains can be used to assess amphi-enterobactin production by cross-feeding assay using HY01 derivatives as sources of anguibactin and amphi-enterobactin. The results in Fig. 4 show that amphi-enterobactin production can be clearly detected from wild-type HY01 that produces both anguibactin and amphi-enterobactin. All controls of anguibactin (HY01∆aebF), amphi-enterobactin (HY01∆angR) and ferric ammonium citrate (FAC) showed expected results, and the fapA mutant was able to be complemented. These results indicate that these indicator strains can be useful for the detection of amphi-enterobactin. Then, we tested amphi-enterobactin production from various V. campbellii and V. harveyi strains using these indicator strains without chloramphenicol supplementation because wild strains of V. campbellii and V. harveyi are sensitive to chloramphenicol. As shown in Fig. 5, amphi-enterobactin production was observed in all strains tested and the mutation in the fapA gene caused negative cross-feeding results, indicating that the cross-feeding occurred specifically due to the presence of FapA. These results demonstrate that amphi-enterobactin is commonly produced from V. campbellii and V. harveyi strains.
Fig. 4

Confirmation of indicator strains that detect amphi-enterobactin but not anguibactin. V. campbellii HY01∆angRaebFfatA(pMMB208), V. campbellii HY01∆angRaebFfatAfapA(pMMB208) and V. campbellii HY01∆angRaebFfatAfapA(pMMB208-fapA) were mixed with melted AB agar supplemented with 1 mM IPTG and 10 µg/mL chloramphenicol. 5 µL of siderophore producers such as anguibactin and amphi-enterobactin producer, V. campbellii HY01(pMMB208); amphi-enterobactin producer, V. campbellii HY01∆angR(pMMB208); anguibactin producer, V. campbellii HY01∆aebF(pMMB208) and 1 µL of 10 mg/mL ferric ammonium citrate (FAC) as a positive control were spotted on the plates and incubated at ambient temperature (approximately 23 °C). Appearance of growth halos around spots was monitored after 16 h incubation until 5 days, and the results were consistent during monitoring. The representative pictures were taken after 40 h incubation

Fig. 5

Amphi-enterobactin production from various V. campbellii and V. harveyi strains. Indicator strains such as V. campbellii HY01∆angRaebFfatA(pMMB208) and V. campbellii HY01∆angRaebFfatAfapA(pMMB208) were mixed with melted AB agar containing a higher amount of Agar Noble (1%) to prevent swarming of some of strains. 5 µL of overnight culture of V. harveyi and V. campbellii strains and 1 µL of 10 mg/mL ferric ammonium citrate (FAC) as a positive control were spotted on the plates and incubated at ambient temperature (approximately 23 °C). Appearance of growth halos around spots was monitored after 16 h incubation until 5 days, and the results were consistent during the monitoring period. The representative pictures were taken after 40 h incubation

We also tested whether amphi-enterobactin can be utilized by V. anguillarum, which is another anguibactin producer, using a V. anguillarum 775 derivative, strain CC9–8, that carries the genes fvtA and fetA but lacks the Fe(III)-anguibactin receptor FatA and has been used as an indicator strain of Fe(III)-enterobactin and Fe(III)-vanchrobactin [28, 30]. As shown in Fig. 6a, the V. anguillarum 775-derived indicator strain CC9–8 that lacks the Fe(III)-anguibactin receptor gene fatA was able to utilize amphi-enterobactin produced by HY01∆angR but not anguibactin produced by HY01∆aebF, indicating that CC9–8 can take up amphi-enterobactin from V. campbellii HY01 while another V. anguillarum 775-derived indicator strain CC9–16∆fvtAfetA that carries the fatA gene but lacks Fe(III)-vanchrobactin/Fe(III)-enterobactin receptors, fvtA and fetA, did not cross-feed with HY01∆angR that only produces amphi-enterobactin. These results indicate that although V. anguillarum 775 does not carry the fapA gene, this strain can utilize Fe(III)-amphi-enterobactin or its break-down products via FvtA and/or FetA. Furthermore, the results in 6b show that CC9–8 can also take up siderophores from all V. campbellii and V. harveyi strains tested, suggesting that V. anguillarum can take up Fe(III)-amphi-enterobactin produced by various V. campbellii and V. harveyi strains. Furthermore, we performed bioassay using single and double mutants of the genes fvtA and fetA constructed in V. anguillarum H775-3, a pJM1-less derivative of strain 775 that lacks the anguibactin system, to clarify which receptor, FvtA and/or FetA is involved in the Fe(III)-amphi-enterobactin transport in V. anguillarum. As shown in Fig. 7, both single fvtA and fetA mutants still can take up amphi-enterobactin produced by V. campbellii HY01∆angR, although the mutation in fvtA showed a dramatic reduction in the amphi-enterobactin transport while the fetA mutant showed a similar growth level as compared with wild-type H775-3. The double fvtA and fetA mutant did not cross-feed with V. campbellii HY01∆angR, and the overexpression of fvtA or fetA in trans in the double ∆fvtAfetA mutant recovered the Fe(III)-amphi-enterobactin transport phenotype. These results indicate that both FvtA and FetA can transport Fe(III)-amphi-enterobactin or the break-down products; these results are similar to our previous report in which we used canonical enterobactin [28].
Fig. 6

V. anguillarum can take up amphi-enterobactin produced by V. campbellii and V. harveyi. The V. anguillarum CC9–8 strain that lacks the Fe(III)-anguibactin specific outer membrane receptor FatA but carries Fe(III)-vanchrobactin/Fe(III)-enterobactin receptors, FvtA and FetA, and CC9–16∆fvtAfetA that carry FatA but lacks FvtA and FetA were used as an indicator strain. The indicator strains were mixed with melted AB agar containing a higher amount of Agar Noble (1%) to prevent swarming of some of strains. 5 µL of overnight culture of V. campbellii HY01 derivatives such as anguibactin [Ang] and amphi-enterobactin [Amphi-ent] producer, V. campbellii HY01(pMMB208); amphi-enterobactin producer, V. campbellii HY01∆angR(pMMB208); anguibactin producer, V. campbellii HY01∆aebF(pMMB208) (a) as well as various V. harveyi and V. campbellii strains (b) were spotted on the plates, and 1 µL of 10 mg/mL ferric ammonium citrate (FAC) was as a positive control in a. Plates were incubated at ambient temperature (approximately 23 °C). Appearance of growth halos around spots was monitored after 16 h incubation until 5 days, and the results were consistent during the monitoring period. The representative pictures were taken after 40 h incubation. The V. harveyi strains used in b did not cross-feed with CC9–16∆fvtAfetA in our previous study [17]

Fig. 7

Fe(III)-amphi-enterobactin can be transported by both FvtA and FetA in V. anguillarum. V. anguillarum H775-3 is a pJM1-cured derivative of strain 775, that lack the anguibactin system but Fe(III)-vanchrobactin/Fe(III)-enterobactin receptors, FvtA and FetA. The fvtA and/or fetA deletion mutants constructed in the H775-3 strain were tested for the utilization of amphi-enterobactin [Amphi-enter] produced by V. campbellii HY01∆angR (pMMB208). Overnight culture of V. anguillarum H775-3 derivatives such as wild-type (pMMB208), ∆fvtA(pMMB208), ∆fetA(pMMB208), ∆fvtAfetA(pMMB208), ∆fvtAfetA(pMMB208-fvtA) and ∆fvtAfetA(pMMB208-fetA) were mixed with melted AB agar supplemented with 1 mM IPTG and 10 µg/mL chloramphenicol. 5 µL of amphi-enterobactin [Amphi-ent] producer V. campbellii HY01∆angR(pMMB208) and 1 µL of 10 mg/mL FAC as a positive control were spotted on the plates and incubated at ambient temperature (approximately 23 °C). Appearance of growth halos around spots was monitored after 16 h incubation until 5 days, and the results were consistent during monitoring. The pictures were taken after 40 h incubation

The presence of amphi-enterobactins was tested by ESI–MS analysis of extracts from various V. campbellii and V. harveyi strains, showing that all strains tested produce a suite of amphi-enterobactin with various fatty acid appendages. Four amphi-enterobactin species (with fatty acids, C10:0, C14:0 OH, C14:0 and C16:1) were newly identified, in addition to seven amphi-enterobactin siderophores we previously identified in V. campbellii BAA-1116 [16] (Table 2). We also detected a suite of ten amphi-enterobactin siderophores from V. campbellii DS40M4 in which we previously reported to produce anguibactin, as well as mono, di- and trivanchrobactin [20]. We also found that the amount of amphi-enterobactins produced varies among different strains.

Anguibactin and amphi-enterobactin systems share the aebA gene that is responsible for 2,3-dihydroxybenzoic acid (DHBA) biosynthesis

2,3-Dihydroxybenzoic acid is a precursor of both anguibactin and amphi-enterobactin. Our previous study suggested that the anguibactin biosynthesis and transport gene cluster lacks DHBA biosynthetic genes, but another siderophore biosynthetic cluster, which we later found to be an amphi-enterobactin biosynthetic cluster, carries the DHBA biosynthetic genes [16, 17, 31]. The localization of DHBA biosynthetic genes outside of the acinetobactin biosynthetic cluster was reported in human pathogen Acinetobacter baumannii [32] and in the fish pathogen Aeromonas salmonicida subspecies salmonicida [33]. In the latter case, acinetobactin shares the DHBA biosynthetic genes located in the siderophore cluster for amonabactin. To investigate whether a similar situation occurs in V. campbellii, one of the DHBA biosynthetic genes, aebA, was mutated in V. campbellii HY01, and the production of DHBA in culture supernatants was tested by Arnow’s assay. As shown in Fig. 8a, the wild-type HY01 strain produced DHBA, while DHBA production was not detected from the culture supernatants of the ∆aebA strain. The production of DHBA was recovered when the aebA gene was expressed in trans in ∆aebA. These results indicate that the aebA gene is essential for DHBA biosynthesis in V. campbellii HY01. Furthermore, the production of anguibactin and amphi-enterobactin was evaluated by bioassay (Fig. 8b). In this experiment, crude extracts prepared from supernatants (by XAD7HP) and cell pellets (by methanol) instead of siderophore-producing bacteria were used in the bioassay to avoid cross-feeding of DHBA that would allow ∆aebA to use DHBA produced by indicator strains to complement ∆aebA. Both anguibactin and amphi-enterobactin production were abolished in the ∆aebA mutant, and the aebA-complemented strain recovered the production of both siderophores, indicating that the aebA gene is essential for the biosynthesis of both anguibactin and amphi-enterobactin.
Fig. 8

The aebA gene involved in the 2,3-dihydroxybenzoic acid (DHBA) biosynthesis is essential for both anguibactin and amphi-enterobactin production. aaebA does not produce DHBA. Culture supernatants of overnight culture of V. campbellii HY01(pMMB208), HY01∆aebA(pMMB208) and HY01∆aebA(pMMB208-aebA) grown in AB broth supplemented with 10 µg/mL chloramphenicol and 1 mM IPTG were used for Arnow’s assay to check the presence of DHBA. The reaction was determined by OD510, and the OD510 values were normalized to the cell density of V. campbellii culture (OD600). Experiments were repeated four times, and the error bars represent standard deviation. b The aebA gene is essential for the biosynthesis of anguibactin as well as amphi-enterobactin. HY01∆angRaebFfapA(pMMB208) and HY01∆angRaebFfatA(pMMB208) are used as indicator strains to detect anguibactin and amphi-enterobactin, respectively. The indicator strains were mixed with melted AB agar supplemented with 1 mM IPTG and 10 µg/mL chloramphenicol. 1 µL of extracts (10 mg/mL) obtained from HY01∆aebA(pMMB208) and HY01∆aebA(pMMB208-aebA) were spotted on bioassay plates, and incubated at ambient temperature (approximately 23 °C). Extracts from anguibactin and amphi-enterobactin producer, HY01(pMMB208); amphi-enterobactin producer, HY01∆angR(pMMB208); anguibactin producer, HY01∆aebF(pMMB208) were used as controls. Appearance of growth halos around spots was monitored after 16 h incubation until 5 days, and the results were consistent during monitoring. The pictures were taken after 40 h incubation

Discussion

Vibrio campbellii and V. harveyi are prevalent in seawater and both are also known to be pathogens of marine vertebrates and invertebrates. V. campbellii BAA-1116 and HY01 (both formerly V. harveyi) produce anguibactin, a well-known siderophore involved in the virulence of the fish pathogen V. anguillarum. Anguibactin was specifically detected by bioassay among V. campbellii strains but not V. harveyi strains following analysis by microarray-based comparative genomic hybridization and multilocus sequence analyses [17].

In this study, we identified and characterized the fapA gene encoding the outer membrane protein FapA that is the only Fe(III)-amphi-enterobactin specific outer membrane receptor in V. campbellii HY01, and determined that FapA also can take up exogenous canonical enterobactin. This result suggests that the FapA protein might not recognize the structural difference between amphi-enterobactin and enterobactin and this could provide flexibility to accept exogenous enterobactin produced by other bacteria, although we cannot exclude the possibility that V. campbellii can take up the break-down products [34] rather than intact cyclic siderophores, as observed in V. cholerae [35]. We also found that V. campbellii HY01 cannot transport vanchrobactin produced by V. anguillarum while amphi-enterobactin is utilized by V. anguillarum. These results provide an example of harsh competition to acquire iron among marine vibrios.

Various strains of V. campbellii and V. harveyi could cross-feed with V. campbellii HY01 under iron limiting conditions, and the fapA gene was responsible for the cross-feeding, confirming that amphi-enterobactin is commonly produced by V. campbellii and V. harveyi strains. We previously showed that the anguibactin biosynthesis and transport cluster are located close to the pheST t-RNA locus (Fig. 2), indicating the possibility of horizontal transfer of this cluster [17]. Furthermore, we previously suggested that the anguibactin system and the amphi-enterobactin system appear to share biosynthesis genes (aebA, B, C and E) for 2,3-dihydroxybenzoic acid, which is a precursor of both siderophores, and the genes are located in the amphi-enterobactin cluster [17, 31]. We here showed that the aebA gene is indeed essential for 2,3-dihydroxybenzoic acid production as well as the production of both siderophores. Based on current and previous results, we hypothesize that the amphi-enterobactin system is a native siderophore present before V. campbellii and V. harveyi were separated during evolution while the anguibactin system was acquired by V. campbellii during or after separation of these species.

We further tested amphi-enterobactin production by chemical approaches. ESI–MS analysis of extracts from various V. campbellii and V. harveyi strains showed that all strains tested produce a suite of amphi-enterobactin with various fatty acid appendages, and the amount of amphi-enterobactin varied among V. campbellii and V. harveyi strains. In the course of our investigation, we found four new amphi-enterobactin species, two of which were recently reported by McRose et al. [34]. These results indicate that V. campbellii and V. harveyi have been going through dynamic evolution in the amphi-enterobactin system with unknown mechanisms possibly to fit to different environmental conditions.

In V. anguillarum, it was shown that acquisition of the pJM1 plasmid encoding anguibactin system silenced the native chromosomally encoding vanchrobactin siderophore system by the transposon originating from the pJM1 plasmid, because anguibactin is a stronger chelator than vanchrobactin [17, 30]. The present study showed that all V. campbellii strains produce amphi-enterobactin in addition to anguibactin, and both siderophores were able to be detected by specific siderophore bioassay. These results suggest that V. campbellii strains are capable of managing anguibactin and amphi-enterobactin to be functional together as opposed to the case of V. anguillarum, and conservation of the amphi-enterobactin system indicates the importance of the system in their life cycle. Amphiphilic siderophores, including amphi-enterobactin, are widely produced by marine bacteria and the amphiphilic properties make these siderophores cell-associated and prevent siderophore diffusion that is a critical aspect of the siderophore system in oceanic environment, [36, 37] while anguibactin is a major virulence factor of a pathogen V. anguillarum [18, 19]. Thus, it is possible that amphi-enterobactin could provide an advantage to V. campbellii and V. harveyi to survive in seawater, while the anguibactin system could be beneficial for some strains to better fit to certain host environments.

Notes

Acknowledgements

The research reported here made use of the shared facilities of the UCSB MRSEC (NSF DMR 1720256). We would like to thank the late Professor Jorge Crosa for his valuable suggestions. This work was supported by NSF CHE-171076 (AB).

Supplementary material

775_2018_1601_MOESM1_ESM.pdf (3.2 mb)
Supplementary material 1 (PDF 3311 kb)

References

  1. 1.
    Crichton R (2016) Iron metabolism: from molecular mechanisms to clinical consequences. Wiley, New YorkCrossRefGoogle Scholar
  2. 2.
    Crosa JH, Walsh CT (2002) Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66:223–249CrossRefGoogle Scholar
  3. 3.
    Crosa JH, Mey AR, Payne SM (2004) Iron transport in bacteria. ASM Press, Washington, D.C.CrossRefGoogle Scholar
  4. 4.
    Owens L, Busico-Salcedo N (2006) Vibrio harveyi: pretty problems in paradise. In: Thompson FL, Austin B, Swings J (eds) The biology of vibrios. ASM Press, Washington, D.C., pp 266–280CrossRefGoogle Scholar
  5. 5.
    Austin B, Zhang X (2006) Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Lett Appl Microbiol 43:119–124CrossRefGoogle Scholar
  6. 6.
    Wang L, Chen Y, Huang H, Huang Z, Chen H, Shao Z (2015) Isolation and identification of Vibrio campbellii as a bacterial pathogen for luminous vibriosis of Litopenaeus vannamei. Aquac Res 46:395–404CrossRefGoogle Scholar
  7. 7.
    Phuoc LH, Corteel M, Nauwynck HJ, Pensaert MB, Alday-Sanz V, Van den Broeck W, Sorgeloos P, Bossier P (2008) Increased susceptibility of white spot syndrome virus-infected Litopenaeus vannamei to Vibrio campbellii. Environ Microbiol 10:2718–2727CrossRefGoogle Scholar
  8. 8.
    Liu J, Zhao Z, Deng Y, Shi Y, Liu Y, Wu C, Luo P, Hu C (2017) Complete genome sequence of Vibrio campbellii LMB 29 isolated from red drum with four native megaplasmids. Front Microbiol 8:2035CrossRefGoogle Scholar
  9. 9.
    Gomez-Gil B, Soto-Rodríguez S, García-Gasca A, Roque A, Vazquez-Juarez R, Thompson FL, Swings J (2004) Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms. Microbiology 150:1769–1777CrossRefGoogle Scholar
  10. 10.
    Lin B, Wang Z, Malanoski AP, O’Grady EA, Wimpee CF, Vuddhakul V, Alves N, Thompson FL, Gomez-Gil B, Vora GJ (2010) Comparative genomic analyses identify the Vibrio harveyi genome sequenced strains BAA-1116 and HY01 as Vibrio campbellii. Environ Microbiol Rep 2:81–89CrossRefGoogle Scholar
  11. 11.
    Ng WL, Bassler BL (2009) Bacterial quorum-sensing network architectures. Annu Rev Genet 43:197–222CrossRefGoogle Scholar
  12. 12.
    Papenfort K, Bassler BL (2016) Quorum sensing signal-response systems in Gram-negative bacteria. Nat Rev Microbiol 14:576–588CrossRefGoogle Scholar
  13. 13.
    Espinoza-Valles I, Vora GJ, Lin B, Leekitcharoenphon P, González-Castillo A, Ussery D, Høj L, Gomez-Gil B (2015) Unique and conserved genome regions in Vibrio harveyi and related species in comparison with the shrimp pathogen Vibrio harveyi CAIM 1792. Microbiology 161:1762–1779CrossRefGoogle Scholar
  14. 14.
    Ke HM, Prachumwat A, Yu CP, Yang YT, Promsri S, Liu KF, Lo CF, Lu MJ, Lai MC, Tsai IJ, Li WH (2017) Comparative genomics of Vibrio campbellii strains and core species of the Vibrio Harveyi clade. Sci Rep 7:41394CrossRefGoogle Scholar
  15. 15.
    Urbanczyk H, Ogura Y, Hayashi T (2013) Taxonomic revision of Harveyi clade bacteria (family Vibrionaceae) based on analysis of whole genome sequences. Int J Syst Evol Microbiol 63:2742–2751CrossRefGoogle Scholar
  16. 16.
    Zane HK, Naka H, Rosconi F, Sandy M, Haygood MG, Butler A (2014) Biosynthesis of amphi-enterobactin siderophores by Vibrio harveyi BAA-1116: identification of a bifunctional nonribosomal peptide synthetase condensation domain. J Am Chem Soc 136:5615–5618CrossRefGoogle Scholar
  17. 17.
    Naka H, Actis LA, Crosa JH (2013) The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid-encoded system of Vibrio anguillarum. MicrobiologyOpen 2:182–194CrossRefGoogle Scholar
  18. 18.
    Crosa JH (1980) A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system. Nature 284:566–568CrossRefGoogle Scholar
  19. 19.
    Naka H, Crosa JH (2011) Genetic determinants of virulence in the marine fish pathogen Vibrio anguillarum. Fish Pathol 46:1–10CrossRefGoogle Scholar
  20. 20.
    Sandy M, Han A, Blunt J, Munro M, Haygood M, Butler A (2010) Vanchrobactin and anguibactin siderophores produced by Vibrio sp. DS40M4. J Nat Prod 73:1038–1043CrossRefGoogle Scholar
  21. 21.
    Soengas RG, Anta C, Espada A, Paz V, Ares IR, Balado M, Rodriguez J, Lemos ML, Jimenez C (2006) Structural characterization of vanchrobactin, a new catechol siderophore produced by the fish pathogen Vibrio anguillarum serotype O2. Tetrahedron Lett 47:7113–7116CrossRefGoogle Scholar
  22. 22.
    Soengas RG, Anta C, Espada A, Nieto RM, Larrosa M, Rodríguez J, Jiménez C (2007) Vanchrobactin: absolute configuration and total synthesis. Tetrahedron Lett 48:3021–3024CrossRefGoogle Scholar
  23. 23.
    Reitz ZL, Sandy M, Butler A (2017) Biosynthetic considerations of triscatechol siderophores framed on serine and threonine macrolactone scaffolds. Metallomics 9:824–839CrossRefGoogle Scholar
  24. 24.
    Thode SK, Rojek E, Kozlowski M, Ahmad R, Haugen P (2018) Distribution of siderophore gene systems on a Vibrionaceae phylogeny: database searches, phylogenetic analyses and evolutionary perspectives. PLoS One 13:e0191860CrossRefGoogle Scholar
  25. 25.
    Taga ME, Xavier KB (2011) Methods for analysis of bacterial autoinducer-2 production. Curr Protoc Microbiol Chapter 1(Unit1C):1Google Scholar
  26. 26.
    Rogers HJ (1973) Iron-binding catechols and virulence in Escherichia coli. Infect Immun 7:445–456PubMedPubMedCentralGoogle Scholar
  27. 27.
    Senanayake SD, Brian DA (1995) Precise large deletions by the PCR-based overlap extension method. Mol Biotechnol 4:13–15CrossRefGoogle Scholar
  28. 28.
    Naka H, Crosa JH (2012) Identification and characterization of a novel outer membrane protein receptor FetA for ferric enterobactin transport in Vibrio anguillarum 775 (pJM1). Biometals 25:125–133CrossRefGoogle Scholar
  29. 29.
    Arnow LE (1937) Colorimetric determination of the components of 3, 4-dihydroxyphenylalanine-tyrosine mixtures. J Biol Chem 118:531–537Google Scholar
  30. 30.
    Naka H, López CS, Crosa JH (2008) Reactivation of the vanchrobactin siderophore system of Vibrio anguillarum by removal of a chromosomal insertion sequence originated in plasmid pJM1 encoding the anguibactin siderophore system. Environ Microbiol 10:265–277PubMedGoogle Scholar
  31. 31.
    Naka H, Liu M, Actis LA, Crosa JH (2013) Plasmid- and chromosome-encoded siderophore anguibactin systems found in marine vibrios: biosynthesis, transport and evolution. Biometals 26:537–547CrossRefGoogle Scholar
  32. 32.
    Penwell WF, Arivett BA, Actis LA (2012) The Acinetobacter baumannii entA gene located outside the acinetobactin cluster is critical for siderophore production, iron acquisition and virulence. PLoS One 7:e36493CrossRefGoogle Scholar
  33. 33.
    Balado M, Souto A, Vences A, Careaga VP, Valderrama K, Segade Y, Rodríguez J, Osorio CR, Jiménez C, Lemos ML (2015) Two catechol siderophores, acinetobactin and amonabactin, are simultaneously produced by Aeromonas salmonicida subsp. salmonicida sharing part of the biosynthetic pathway. ACS Chem Biol 10:2850–2860CrossRefGoogle Scholar
  34. 34.
    McRose DL, Baars O, Seyedsayamdost MR, Morel FMM (2018) Quorum sensing and iron regulate a two-for-one siderophore gene cluster in Vibrio harveyi. Proc Natl Acad Sci USA (in press).  https://doi.org/10.1073/pnas.1805791115 CrossRefGoogle Scholar
  35. 35.
    Wyckoff EE, Allred BE, Raymond KN, Payne SM (2015) Catechol siderophore transport by Vibrio cholerae. J Bacteriol 197:2840–2849CrossRefGoogle Scholar
  36. 36.
    Sandy M, Butler A (2009) Microbial iron acquisition: marine and terrestrial siderophores. Chem Rev 109:4580–4595CrossRefGoogle Scholar
  37. 37.
    Vraspir JM, Butler A (2009) Chemistry of marine ligands and siderophores. Ann Rev Mar Sci 1:43–63CrossRefGoogle Scholar

Copyright information

© SBIC 2018

Authors and Affiliations

  • Hiroaki Naka
    • 1
  • Zachary L. Reitz
    • 2
  • Aneta L. Jelowicki
    • 2
  • Alison Butler
    • 2
  • Margo G. Haygood
    • 1
  1. 1.Department of Medicinal Chemistry, L.S. Skaggs Pharmacy InstituteUniversity of UtahSalt Lake CityUSA
  2. 2.Department of Chemistry and BiochemistryUniversity of CaliforniaSanta BarbaraUSA

Personalised recommendations