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Appl Environ Microbiol. 2010 April; 76(8): 2425–2432.
Published online 2010 February 26. doi:  10.1128/AEM.03066-09
PMCID: PMC2849222

Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1[down-pointing small open triangle]

Abstract

Shewanella oneidensis MR-1 respires a wide range of anaerobic electron acceptors, including sparingly soluble Fe(III) oxides. In the present study, S. oneidensis was found to produce Fe(III)-solubilizing organic ligands during anaerobic Fe(III) oxide respiration, a respiratory strategy postulated to destabilize Fe(III) and produce more readily reducible soluble organic Fe(III). In-frame gene deletion mutagenesis, siderophore detection assays, and voltammetric techniques were combined to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration were synthesized via siderophore biosynthesis systems and (ii) if the Fe(III)-siderophore reductase was required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. Genes predicted to encode the siderophore (hydroxamate) biosynthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) were identified in the S. oneidensis genome, and corresponding in-frame gene deletion mutants were constructed. ΔSO3031 was unable to synthesize siderophores or produce soluble organic Fe(III) during aerobic respiration yet retained the ability to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. ΔSO3034 retained the ability to synthesize siderophores during aerobic respiration and to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. These findings indicate that the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are not synthesized via the hydroxamate biosynthesis system and that the Fe(III)-hydroxamate reductase is not essential for respiration of Fe(III)-citrate or Fe(III)-nitrilotriacetic acid (NTA) as an anaerobic electron acceptor.

Bacterial electron transfer to sparingly soluble electron acceptors is a critical component of a wide variety of environmental and energy-generating processes, including biogeochemical cycling of metals, degradation of natural and contaminant organic matter, weathering of clays and minerals, biomineralization of Fe-bearing minerals, reductive precipitation of toxic metals and radionuclides, and electricity generation in microbial fuel cells (17, 33, 34). Anaerobic and facultatively anaerobic bacteria capable of respiring sparingly soluble (<10−25 M at pH 7) Fe(III) oxides are ubiquitous in nature and may be found in marine, freshwater, and terrestrial environments, including metal- and radionuclide-contaminated subsurface aquifers (25, 34). Fe(III)-respiring prokaryotes are also deeply rooted and scattered throughout the domains Bacteria and Archaea (possibly indicating an ancient metabolic process) and include hyperthermophiles, psychrophiles, acidophiles, and extreme barophiles (34). Despite their potential environmental, energy-generating, and evolutionary significance, the molecular details of microbial Fe(III) respiration remain unclear.

Fe(III)-respiring, neutrophilic bacteria are presented with a unique physiological challenge: they are required to respire anaerobically on electron acceptors found largely as sparingly soluble Fe(III) oxides presumably unable to contact periplasm- or inner membrane (IM)-localized electron transport systems. To overcome this problem, Fe(III)-respiring bacteria are postulated to employ novel respiratory strategies not found in other bacteria (e.g., aerobes, denitrifiers, sulfate-reducing bacteria, and methanogens) that respire soluble electron acceptors (17, 38). The novel respiratory strategies include (i) a direct-contact pathway in which terminal Fe(III) reductases are secreted to the cell outer membrane (OM), where they contact and deliver electrons directly to external Fe(III) oxides (18, 23, 40, 42, 48, 57, 64, 67), (ii) a two-step electron shuttling pathway in which bacterially reduced endogenous or exogenous electron shuttles deliver electrons to external Fe(III) oxides in a second (abiotic) electron transfer reaction (11, 26, 39, 45), and (iii) a two-step Fe(III) chelation (solubilization) pathway in which Fe(III) oxides are first nonreductively dissolved by endogenously synthesized organic ligands prior to reduction of the resulting soluble organic Fe(III) [Fe(III) bound to an organic molecule] complexes (36, 59).

Candidate organic ligands for production of soluble organic Fe(III) during anaerobic Fe(III) oxide respiration include siderophores, the Fe(III)-chelating compounds synthesized and secreted by a wide variety of bacteria and fungi for solubilization and subsequent assimilation of otherwise inaccessible Fe(III) substrates (12, 44, 49, 63). Hydroxamate-type siderophores are produced via N6 hydroxylation and N6 acylation of l-ornithine and, in some cases, cyclization to macrocyclic ring structures (13). The macrocyclic siderophores bisucaberin and putrebactin, for example, are two structural analogs of the cyclic bis(hydroxamate) siderophore alcaligin, synthesized by Aliivibrio salmonicida and Shewanella putrefaciens strain 200, respectively (27, 32, 65). After transport across the cell envelope via a TonB-dependent pathway, Fe(III) is subsequently released from the Fe(III)-siderophore complex by ligand exchange reactions promoted by siderophore ligand hydrolysis and/or protonation or by Fe(III)-siderophore reduction and release of Fe(II) to acceptor ligands (9, 66).

The main objectives of the present study were to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are synthesized by Fe(III)-siderophore biosynthesis systems and (ii) if Fe(III)-siderophore reductases are required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. The experimental strategy for this study included (i) identification of genes encoding the siderophore biosynthesis and Fe(III)-siderophore reductase systems in the S. oneidensis genome, (ii) generation of in-frame deletions in the corresponding siderophore biosynthesis and Fe(III)-siderophore reductase genes, (iii) tests of the resulting siderophore biosynthesis mutants for production of siderophores and soluble organic Fe(III) during aerobic and anaerobic Fe(III) oxide respiration, and (iv) tests of the resulting Fe(III)-siderophore reductase mutants for respiration of soluble organic Fe(III) as an anaerobic electron acceptor.

MATERIALS AND METHODS

Growth media and cultivation conditions.

All bacterial strains and plasmids used in this study are listed in Table Table1.1. For genetic manipulations, S. oneidensis MR-1 was cultured at 30°C in Luria-Bertani (LB) medium (10 g liter−1 NaCl, 5 g liter−1 yeast extract, 10 g liter−1 tryptone). For anaerobic-growth experiments, cells were cultured in a defined salts medium (SM) (16) supplemented with lactate (18 mM) or formate (30 mM) as a carbon/energy source under a nitrogen atmosphere. When required, antibiotics were added at the following final concentrations: for gentamicin (Gm), 15 μg ml−1, and for chloramphenicol, 25 μg ml−1. For growth of Escherichia coli β2155 λ pir (12), diaminopimelate (DAP) was added at a final concentration of 100 μg ml−1. 2,2′-Dipyridyl experiments were carried out with liquid SM amended with 2,2′-dipyridyl at a range of concentrations (0 to 200 μM) under aerobic growth conditions. Aerobic growth was monitored spectrophotometrically by measuring changes in optical density at 600 nm.

TABLE 1.
Strains and plasmids used in this study

Nucleotide and amino acid sequence analyses.

S. oneidensis genome sequence information was obtained from the Comprehensive Microbial Resource (J. Craig Venter Institute [http://cmr.jcvi.org]). Proteins displaying sequence similarity to SO3030 to SO3034 were identified via BLASTp, available at the National Center for Biotechnology Information (NCBI) (1). Conserved protein domains were identified with Pfam (http://pfam.sanger.ac.uk/) (21) and Prosite (http://www.expasy.org/prosite/) (28) software. Multiple sequence alignments were performed with ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

In-frame gene deletion mutagenesis.

Genes encoding SO3031 and SO3034 were deleted in-frame from the S. oneidensis genome as described previously (8). Regions corresponding to ~750 bp upstream and downstream of each open reading frame (ORF) were PCR amplified with iProof ultrahigh-fidelity polymerase (Bio-Rad, Hercules, CA), generating fragments F1 and F2, which were fused by overlap extension PCR to generate fragment F3. The primers used for construction of ΔSO3031 and ΔSO3034 are listed in Table Table2.2. Fragment F3 was cloned into pKO2.0 with BamHI and SalI restriction endonucleases and electroporated into E. coli strain β2155 λ pir. pKO2.0-F3 was mobilized into recipient S. oneidensis MR-1 via biparental mating procedures (15). A plasmid integrant was identified via PCR analysis, and the mutation was resolved on LB agar containing sucrose (10%). Transconjugants were identified on SM agar containing Gm and confirmed via PCR. Following counterselection on SM agar containing sucrose (10% [wt/vol]), the corresponding in-frame deletion mutant strains (designated ΔSO3031 and ΔSO3034) were isolated and confirmed via PCR (see Fig. S1 in the supplemental material) and DNA sequencing (University of Nevada, Reno Genomics Facility).

TABLE 2.
Primers used in this study

Knock-in complementation analysis.

ΔSO3031 and ΔSO3034 were each complemented via a knock-in complementation strategy similar to that followed in the in-frame gene deletion protocol described above. Wild-type copies of SO3031 and SO3034 were PCR amplified from wild-type S. oneidensis genomic DNA with SO3031- and SO3034-specific primers 3031C1 and 3031C2 and SO3034D1 and SO3034D4, respectively (Table (Table2).2). The resulting amplicon contained the entire ORF and ~750 bp of upstream and downstream DNA for subsequent recombination into ΔSO3031 and ΔSO3034. The amplicons were cloned into pKO2.0 using identical restriction sites, and the resulting constructs were subsequently transformed into E. coli strains as described above. Knock-in complementation was performed as described for in-frame deletion above, with the exception that ΔSO3031 and ΔSO3034 were used as recipient strains to generate the corresponding knock-in complemented strains ΔSO3031-KI and ΔSO3034-KI, respectively. Insertion into the proper locations of the ΔSO3031-KI and ΔSO3034-KI genomes was confirmed via PCR amplification with flanking primers SO3031DTF and SO3031DTR for ΔSO3031 and SO3034DTF and SO3034DTR for ΔSO3034 (Table (Table2;2; see also Fig. S1 in the supplemental material), followed by DNA sequencing (University of Nevada, Reno Genomics Center).

Determination of the overall respiratory capability of the S. oneidensis wild-type and ΔSO3031 and ΔSO3034 mutant strains.

The S. oneidensis wild-type and ΔSO3031 and ΔSO3034 mutant strains were inoculated in liquid SM (initial concentration of 107 cells ml−1) amended with either 18 mM lactate or 30 mM formate as an electron donor and either O2, 15 mM nitrate, 50 mM dimethyl sulfoxide (DMSO), 25 mM trimethylamine-N-oxide (TMAO), 10 mM fumarate, 10 mM thiosulfate, 50 mM Fe(III)-citrate, colloidal 10 mM MnO2, 10 mM Mn(III)-pyrophosphate, 20 mM Fe(III)-desferrioxamine B [Fe(III)-DEFB], 20 mM Fe(III)-nitrilotriacetic acid [Fe(III)-NTA], or 40 mM hydrous Fe(III)-oxide (HFO) as an electron acceptor (3, 43, 46, 54, 61). For aerobic growth, compressed air was vigorously bubbled through the reactors. Anaerobic conditions were maintained by continuous sparging with N2(g). Cell growth was monitored by measuring absorbance at 600 nm over time. Nitrite (NO2) was measured by diluting samples 250-fold in a solution consisting of 9.6 mM sulfanilic acid, 96 mM KHSO4, and 3.2 mM N,N-ethylenediamine (41). Samples were held in the dark for 15 min prior to absorbance measurements at 510 nm. Mn(III) reduction was monitored colorimetrically by filtering samples through a disposable nylon filter with a pore size of 0.22 μm (Millex) and subsequently monitoring absorbance spectrophotometrically at 480 nm (31). Mn(IV) reduction was monitored by mixing samples at a 1:9 ratio with 2 mM benzidine in a 10% acetic acid solution and monitoring absorbance spectrophotometrically at 424 nm (7, 14). Fe(III) reduction rates were determined by monitoring Fe(II) production over time via HCl extraction, followed by a Ferrozine colorimetric assay (35, 58). Cell growth was monitored by direct cell counts of acridine orange-stained cells via epifluorescence microscopy (Carl Zeiss AxioImager Z1 microscope) (8, 14).

Siderophore detection via CAS-based techniques.

Siderophores were detected during growth on liquid or solid SM via application of chrome azurol-S (CAS)-based techniques. CAS screening plates were prepared using a modified version of a previously described procedure (56). Blue CAS (Sigma Aldrich) agar was prepared by adding 60.5 mg of CAS dye dissolved in 50 ml water to 10 ml of an acidic solution of FeCl3 (1 mM FeCl3, 10 mM HCl). This mixture was slowly added to 40 ml of a 0.2 mM solution of hexadecyltrimethylammonium (HDTMA) (Sigma Aldrich), and the resulting solution was autoclaved, cooled to 55°C, and added to 900 ml sterile SM supplemented with 1.5% (wt/vol) agar (CAS agar). CAS shuttling solution was prepared as previously described (56). Siderophore production by the S. oneidensis wild-type and ΔSO3031 and ΔSO3034 mutant strains was monitored by patching colonies onto CAS agar plates, incubating the colonies aerobically for 24 h, and visually scoring the colony periphery for yellow halos. Siderophore production was also monitored during aerobic growth in liquid SM with lactate as an electron donor. Samples were centrifuged for 1 min (12,000 × g), and the resulting supernatant was mixed with 0.5 ml CAS shuttling solution and incubated for 3 h. Samples were subsequently measured spectrophotometrically at 630 nm to infer siderophore concentration.

Under anaerobic Fe(III) oxide-respiring growth conditions, a modified CAS assay was employed to detect production of Fe(III)-chelating organic ligands. In the modified CAS assay, aliquots from anaerobic Fe(III) oxide-respiring cultures were withdrawn and centrifuged at 12,000 × g for 1 min, and the resulting supernatant reacted with Fe(III)-free CAS shuttling solution for 6 h prior to measurement of Fe(III)-CAS complex formation colorimetrically at 630 nm. The modified CAS assay facilitates detection of organic ligands with Fe(III)-binding affinities that are lower than those observed with traditional siderophores [the traditional CAS assay detects a ligand exchange reaction between Fe(III)-CAS and a competing siderophore]. Production of catecholate-type siderophores was determined with the Arnow assay by reacting culture supernatants with equal volumes of 0.5 M HCl, a solution of 10% (wt/vol) sodium nitrate and 10% (wt/vol) sodium molybdate, and 1.0 M NaOH. The presence of catecholate ligands was determined spectrophotometrically at 505 nm (2).

Detection of soluble organic Fe(III) via voltammetric analyses.

The S. oneidensis wild-type and ΔSO3031 and ΔSO3034 mutant strains were incubated in 100-ml PEEK batch reactors containing liquid SM supplemented with 30 mM HFO as an electron acceptor and 20 mM sodium lactate as an electron donor. Reactors were inoculated with cells at an initial concentration of 2 × 107 cells ml−1 and incubated in a Coy chamber under anaerobic conditions (atmosphere of 85% N2, 10% CO2, 5% H2) with gentle mixing. An aliquot from each vessel was extracted in 0.5 M HCl for total Fe(II) measurement via the Ferrozine colorimetric method (35, 58). Soluble organic Fe(III) was measured using voltammetric microelectrodes and a computer-operated DLK-100A potentiostat (Analytical Instrument Systems, Inc.) (59). Electrodes for voltammetric analysis consisted of 100-μm-diameter gold-mercury (Au-Hg) amalgam working electrodes and 0.5-mm-diameter Ag-AgCl reference and platinum counterelectrodes. Working electrodes were polished, plated, and conditioned as previously described (5). The voltammetric conditions used in triplicate cathodic square wave voltammetry (CSWV) measurements included preconditioning at −0.9 V for 10 s and deposition at −0.1 V for 10 s to concentrate Fe(III) species at the electrode surface. A scan rate of 200 mV s−1 from −0.1 to −1.8 V with a pulse height of 0.05 V was used as previously described (10, 59, 60, 62). Electrodes were calibrated for Mn2+ (minimum detection limit [MDL], ~5 μmol liter−1) by CSWV in degassed medium, and the pilot ion method was used to quantify concentrations of Fe(II). Voltammograms were integrated using a semiautomated software program developed for these applications (6). Initial rates of soluble organic Fe(III) production were determined by linear regression of the increase in soluble organic Fe(III) current intensities for 24 h (disregarding any initial lag period). Initial rates of Fe(III) reduction were determined by linear regression of the total Fe(II) production rate during the period of soluble organic Fe(III) production. All rates were normalized to the cellular protein content determined via the Bradford assay (4).

RESULTS

Identification of S. oneidensis genes encoding the hydroxamate biosynthesis system, the Fe(III)-hydroxamate receptor, and the Fe(III)-hydroxamate reductase.

Genome-wide sequence analysis identified an S. oneidensis gene cluster (SO3030 to SO3034) whose predicted proteins displayed high homology to enzymes carrying the hydroxamate synthesis system, the Fe(III)-hydroxamate receptor, and the Fe(III)-hydroxamate reductase of other Gram-negative gammaproteobacteria (Table (Table3).3). Genes encoding synthesis systems for production of catecholate- and α-hydroxycarboxylate-type siderophores were not detected (data not shown). S. oneidensis proteins SO3030 to SO3033 displayed high similarities (64 to 75%) to and correspondingly low E values (0.0 to 10−41) for the lysine-N6-hydroxylase (SO3030), the acyl-coenzyme A (CoA)-N-acyl-transferase (SO3031), the nonribosomal peptide synthase (SO3032), and the TonB-dependent, hydroxamate-type Fe(III)-siderophore receptor (SO3033) found in the gene cluster comprising LF11237 to LF11240 of A. salmonicida (Table (Table3).3). S. oneidensis SO3034 displayed moderate similarity (41%) to and a moderate E value (10−20) for the E. coli FhuF-like Fe(III)-hydroxamate reductase of Enterobacter sp. strain 638 (Table (Table33).

TABLE 3.
Analysis of homologs of S. oneidensis SO3030 to SO3034

The gene cluster comprising SO3030 to SO3034 of S. oneidensis also displayed high similarity (75 to 93%) to and corresponding E values (0.0 to 10−72) for a gene cluster identified in the unannotated genome of S. putrefaciens strain 200 (see Table S1 in the supplemental material), a phylogenetically related strain known to produce the macrocyclic (dihydroxamate-type) siderophore putrebactin (32) and to produce soluble organic Fe(III) during anaerobic Fe(III) oxide respiration (59). Additional genome-wide sequence analyses indicated that proteins with moderate-to-high similarity to the S. oneidensis gene cluster comprising SO3030 to SO3034 were present in all recently sequenced Shewanella genomes, including those of S. putrefaciens CN32, S. putrefaciens W3-18-1, S. amazonensis SB2B, S. denitrificans OS217, S. baltica OS195, S. frigidimarina NCIMB400, S. pealeana ATCC 700345, S. woodyi ATCC 51908, Shewanella sp. strain ANA-3, Shewanella sp. strain MR-4, Shewanella sp. strain MR-7, S. loihica PV-4, S. halifaxens, S. piezotolerans, S. benthica, and S. sediminis (Table (Table33).

Siderophore production under aerobic and anaerobic growth conditions.

Under aerobic growth conditions (and in the absence of 2,2′-dipyridyl), ΔSO3034, ΔSO3031-KI, and ΔSO3034-KI strains produced CAS-reactive siderophores at wild-type rates (Fig. (Fig.1C).1C). ΔSO3031, on the other hand, was unable to produce siderophores under aerobic growth conditions (in the absence of 2,2′-dipyridyl). Aerobic incubations on CAS-supplemented agar plates demonstrated that all strains except ΔSO3031 produced a yellow halo around the colony periphery, an indication that only ΔSO3031 lacked the ability to produce a diffusible, Fe(III)-chelating compound (presumably hydroxamate) that outcompeted CAS for Fe(III) (Fig. (Fig.2).2). Arnow-based assays revealed that none of the strains (including the wild type) produced catecholate-type siderophores at detectable levels during aerobic growth (in the absence of 2,2′-dipyridyl) (data not shown). Under anaerobic Fe(III)-oxide respiring growth conditions, all strains (including ΔSO3031) produced CAS-reactive Fe(III)-chelating ligands at wild-type rates (detected via the modified CAS assay) (Fig. (Fig.1D1D).

FIG. 1.
Siderophore production and Fe(III) reduction. Results are shown for the wild-type (•), ΔSO3031 (○), ΔSO3034 ([filled triangle]), ΔSO3031-KI ([open triangle]), and ΔSO3034-KI ([filled square]) strains. (A) Soluble Fe(III) production ...
FIG. 2.
Determination of siderophore production via the CAS assay. Wild-type, ΔSO3031, and ΔSO3034 colonies patched onto SM-CAS agar plates and grown aerobically for 24 h. Clearing zones around the patch periphery indicate production of Fe(III)-chelating ...

Overall respiratory capability of the S. oneidensis wild-type and ΔSO3031 and ΔSO3034 mutant strains.

The genes encoding SO3031 and SO3034 were deleted in-frame, and the resulting mutants (ΔSO3031 and ΔSO3034, respectively) were tested for aerobic and anaerobic growth on a suite of 11 electron acceptors. ΔSO3031 and ΔSO3034 retained the ability to grow at wild-type rates on all electron acceptors (with lactate or formate as an electron donor) (see Fig. S2 in the supplemental material), including sparingly soluble Fe(III) oxides (HFO) and soluble organic Fe(III) [supplied as Fe(III)-citrate and Fe(III)-NTA] (Fig. (Fig.11 and and3).3). Wild-type S. oneidensis was unable to respire anaerobically on soluble organic Fe(III) supplied as Fe(III)-DEFB (Fig. (Fig.3).3). To differentiate the aerobic-growth deficiency of ΔSO3031 from that of wild-type S. oneidensis, the Fe(II)-scavenging compound 2,2′-dipyridyl was added to SM prior to inoculation. At 2,2′-dipyridyl concentrations of >200 μM, neither ΔSO3031, ΔSO3034, nor the wild-type strain was capable of aerobic growth (data not shown), an indication that Fe was scavenged below the threshold Fe levels required for aerobic growth of all strains, including the wild type. While ΔSO3031 was unable to grow aerobically in the presence of 2,2′-dipyridyl concentrations of 100 μM, the wild-type, ΔSO3034, ΔSO3031-KI, and ΔSO3034-KI strains grew aerobically at normal rates (Fig. (Fig.44).

FIG. 3.
Soluble organic Fe(III) reduction. Fe(II) concentration in incubations with 50 mM Fe(III)-citrate (wild type [•], ΔSO3031 [○], and ΔSO3034 [[filled triangle]]), 20 mM Fe(III)-NTA (wild type [♦], ΔSO3031 [[open diamond]], ...
FIG. 4.
Mutant growth capabilities in the presence of 2,2′ dipyridyl. Results are shown for the wild-type (•), ΔSO3031 (○), ΔSO3034 ([filled triangle]), ΔSO3031-KI ([open triangle]), and ΔSO3034-KI ([filled triangle]) strains. ...

Voltammetric detection of soluble organic Fe(III) under aerobic and anaerobic Fe(III) oxide-respiring growth conditions.

Voltammetric analyses indicated that both ΔSO3031 and ΔSO3034 strains produced soluble organic Fe(III) complexes (at wild-type rates corresponding to 0.03 nA mg protein−1 h−1) and reduced Fe(III) oxides at wild-type rates (500 μmol mg protein−1 h−1) during anaerobic growth on Fe(III) oxides as an electron acceptor (Fig. 1A and B). The S. oneidensis wild-type, ΔSO3031, and ΔSO3034 strains, on the other hand, produced soluble organic Fe(III) at or below detection limits during aerobic growth in the presence of Fe(III) oxides (Fig. (Fig.1A).1A). Neither soluble organic Fe(III) nor Fe(II) was detected in abiotic or heat-killed controls under aerobic or anaerobic conditions (data not shown).

DISCUSSION

Previous voltammetric analyses indicated that S. putrefaciens strain 200 produced soluble organic Fe(III) during anaerobic respiration of sparingly soluble Fe(III) oxides, including hematite, goethite, and 2-line ferrihydrite (59). The results of the present study expand the range of Shewanella species capable of producing soluble organic Fe(III) during anaerobic Fe(III) oxide respiration to include S. oneidensis. Soluble organic Fe(III) production may therefore be a requisite intermediate step in anaerobic respiration of Fe(III) oxides by metal-respiring members of the Shewanella genus. Previous studies indicated that a main pathway for Fe(III) oxide respiration by S. oneidensis entailed direct contact between Fe(III) oxides and cell surface-exposed c-type cytochromes such as MtrC and OmcA (20, 37, 67). Subsequent electrochemical analyses revealed that a distance of <15 Å was required for electron transfer from the catalytic heme group of OmcA to hematite (30). The direct-contact mechanism was recently brought into question, however, by kinetic analyses which demonstrated that OmcA and MtrC are kinetically competent to account for whole-cell reduction of soluble-organic Fe(III) but not for sparingly soluble Fe(III) oxides (51). Fe(III) oxide respiratory pathways that include an intermediate step of nonreductive Fe(III) dissolution may provide several distinct advantages over the direct-contact pathway. Soluble organic Fe(III) produced during nonreductive Fe(III) dissolution may display (i) higher reduction potentials to conserve more energy (59, 69), (ii) lower levels of activation energy to increase reduction rates (59, 64), (iii) higher Fe(II)-binding affinities to prevent Fe(II) passivation of Fe(III) oxide surfaces (50, 52), or (iv) higher solubility to facilitate uptake and reduction by periplasm- or IM-localized terminal Fe(III) reductases (19, 47, 53).

Soluble organic Fe(III) production by Fe(III) oxide-respiring S. putrefaciens strain 200 was hypothesized to entail Fe(III) destabilization by bacterially produced organic ligands with high Fe(III)-chelating capability (59). Siderophores constitute a major class of bacterially produced compounds with high Fe(III)-chelating capability (with conditional stability constants [log K values] ranging from 10 to 60) (29, 63). Several lines of evidence indicate that S. oneidensis synthesizes hydroxamate-like siderophores. Similar to the structurally analogous siderophore bisucaberin (log K = 32), synthesized by A. salmonicida (65), the macrocyclic (dihydroxamate) siderophore putrebactin is synthesized by S. putrefaciens strain 200 (32). Correspondingly, a contiguous five-gene cluster predicted to encode an A. salmonicida-like hydroxamate synthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) was identified in the genomes of both S. oneidensis and S. putrefaciens strain 200 (which displayed nearly identical sequences).

The results of CAS-based siderophore detection assays confirmed that wild-type S. oneidensis produces siderophores during aerobic growth but that ΔSO3031 (harboring an in-frame deletion of the predicted acyl-CoA-N6-acyl transferase) was unable to produce siderophores during aerobic growth. The S. oneidensis wild-type and ΔSO3031 and ΔSO3034 mutant strains retained the ability to grow aerobically or anaerobically on a suite of 11 electron acceptors without addition of exogenous Fe for assimilatory purposes. Aerobic growth of ΔSO3031 was impaired only if the Fe(II)-scavenging compound 2,2′-dipyridyl was supplied at 100 μM levels (the aerobic-growth rates of the wild-type and ΔSO3034 strains were not affected at this level). Background Fe levels in SM [below the detection limit of 0.1 μM Fe(II)] were evidently high enough to sustain aerobic growth of ΔSO3031 even in the absence of the Fe(III)-hydroxamate biosynthesis system. Such Fe-scavenging capability was also recently reported in a study of the ferric uptake regulator (Fur) regulon of S. oneidensis (68). In that study, addition of 2,2′-dipyridyl was also required to differentiate the aerobic-growth rates of the S. oneidensis wild-type and fur mutant strains. In contrast to what was observed under aerobic growth conditions, voltammetric and CAS-based analyses demonstrated that ΔSO3031 produced soluble organic Fe(III) under anaerobic Fe(III) oxide-respiring conditions. Since the hydroxamate biosynthesis synthesis system was disabled in ΔSO3031, this finding indicates that the Fe(III)-chelating ligands produced for anaerobic Fe(III) respiration were not synthesized by the hydroxamate system. Genes encoding other siderophore synthesis systems were not detected in the S. oneidensis genome, an indication that the Fe(III)-chelating ligands produced during anaerobic Fe(III) oxide respiration are synthesized by an as-yet-unidentified biosynthetic pathway.

Previous studies demonstrated that purified complexes of MtrC and OmcA displayed soluble organic Fe(III) reductase activity (57). Production of soluble organic Fe(III) may therefore aid surface-exposed MtrC and/or OmcA in contacting Fe(III) substrates. Alternatively, soluble Fe(III) may diffuse into the periplasm for reduction by other terminal Fe(III) reductases (e.g., MtrA [47, 55]). In the present study, ΔSO3034 also retained the ability to respire Fe(III)-citrate and Fe(III)-NTA at wild-type rates, an indication that the Fe(III)-hydroxamate reductase was not essential for Fe(III)-citrate or Fe(III)-NTA respiration and was most likely dedicated to Fe(III) assimilation. The inability of wild-type S. oneidensis to respire Fe(III) chelated by the trihydroxamate ligand DEFB (log K = 31 [22]) as an anaerobic electron acceptor corroborated this finding. S. putrefaciens strain 200 had previously displayed a similar pattern of soluble organic Fe(III) respiratory capability (24). S. putrefaciens strain 200 reduced Fe(III)-citrate and Fe(III)-NTA (log K = 11 and 16, respectively) but was unable to reduce soluble organic Fe(III) substrates with log K values of >18 (24). This pattern of soluble organic Fe(III) reduction activity may indicate that metal-reducing Shewanella strains respire only soluble organic Fe(III) complexes with log K values less than a threshold level of approximately 18. When this strategy is followed, metal-reducing Shewanella strains may differentiate soluble organic Fe(III) complexes respired as an anaerobic electron acceptor from those assimilated for nutrient purposes. Current work is focused on application of complementary genetic, biochemical, and electrochemical techniques to identify the Fe(III)-chelating ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration and to identify the terminal Fe(III) reductases that transfer electrons to the resulting soluble organic Fe(III) complexes.

Supplementary Material

[Supplemental material]

Acknowledgments

Financial support for this study was provided by the Department of Energy and the National Science Foundation.

Footnotes

[down-pointing small open triangle]Published ahead of print on 26 February 2010.

Supplemental material for this article may be found at http://aem.asm.org/.

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