Thomas J. DiChristina

Thermodynamics of U(VI) sorption onto Shewanellaputrefaciens

Abstract We have conducted acid–base potentiometric titrations and U(VI) sorption experiments using the Gram negative, facultatively anaerobic bacterium Shewanella putrefaciens . Results of reversed titration studies on live, inactive bacteria indicate that their pH-buffering properties result from the equilibrium ionization of three discrete populations of functional groups. Carboxyl ( p K a =5.16±0.04), phosphoryl ( p K a =7.22±0.15) and amine ( p K a =10.04±0.67) groups most likely represent these three resolvable functionalities, based on their p K a values. Site densities for carboxyl, phosphoryl and amine groups on the bacterial surface were approximately 31.7 μmol sites/g bacteria (0.35±0.02 sites/nm 2 ), 8.95 μmol/g (0.11±0.007 sites/nm 2 ) and 38.0 μmol/g (0.42±0.008 sites/nm 2 ), respectively, based on an estimated bacterial specific surface area of 55 m 2 /g. Sorption experiments showed that U(VI) can reversibly complex with the bacterial surface in the pH 2–8 interval, with maximum adsorption occurring at a pH of ∼5. Sorption is not strongly sensitive to ionic strength (NaCl) in the range 0.02–0.10 M. The pH and ionic strength dependence of U(VI) sorption onto S. putrefaciens is similar to that measured for metal-oxide surfaces and Gram positive bacteria, and appears to be similarly governed by competitive speciation constraints. Measured U(VI) sorption is accounted for by using two separate adsorption reactions forming the surface complexes >COO–UO 2 + and >PO 4 H–UO 2 (OH) 2 . Using S. putrefaciens as a model organism for dissimilatory metal-reducing Gram negative anaerobes, our results extend the applicability of geochemical speciation models to include bacteria that are capable of reductively solubilizing or precipitating a wide variety of environmentally and geologically important metals and metallic species.

Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene

Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468-1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).

Seasonal oscillation of microbial iron and sulfate reduction in saltmarsh sediments (Sapelo Island, GA, USA)

Seasonal variations in anaerobic respiration pathways were investigated at three saltmarsh sites using chemical data, sulfate reduction rate measurements, enumerations of culturable populations of anaerobic iron-reducing bacteria (FeRB), and quantification of in situ 16S rRNA hybridization signals targeted for sulfate-reducing bacteria (SRB). Bacterial sulfate reduction in the sediments followed seasonal changes in temperature and primary production of the saltmarsh, with activity levels lowest in winter and highest in summer. In contrast, a dramatic decrease in the FeRB population size was observed during summer at all sites. The collapse of FeRB populations during summer was ascribed to high rates of sulfide production by SRB, resulting in abiotic reduction of bioavailable Fe(III) (hydr)oxides. To test this hypothesis, sediment slurry incubations at 10, 20 and 30 °C were carried out. Increases in temperature and labile organic carbon availability (acetate or lactate additions) increased rates of sulfate reduction while decreasing the abundance of culturable anaerobic FeRB. These trends were not reversed by the addition of amorphous Fe(III) (hydr)oxides to the slurries. However, when sulfate reduction was inhibited by molybdate, no decline in FeRB growth was observed with increasing temperature. Addition of dissolved sulfide adversely impacted propagation of FeRB whether molybdate was added or not. Both field and laboratory data therefore support a sulfide-mediated limitation of microbial iron respiration by SRB. When total sediment respiration rates reach their highest levels during summer, SRB force a decline in the FeRB populations. As sulfate reduction activity slows down after the summer, the FeRB are able to recover.

Microbially Catalyzed Dissolution of Iron and Aluminum Oxyhydroxide Mineral Surface Coatings

Abstract This experimental study investigated the processes by which microbes interact with oxyhydroxide mineral surface coatings using an approach designed to better represent the conditions of natural subsurface environments. The interactions of Shewanella putrefaciens, a facultative anaerobe capable of dissimilatory iron reduction, with coatings of Fe3+ and Al3+ oxyhydroxides on natural quartz and silica glass surfaces were examined. Using synthetic groundwater solutions having- compositions that simulated a typical aquifer, bacteria were seeded onto mineral surfaces (and coatings) and incubated in parallel with abiotic controls for up to 96 h under aerobic and anaerobic conditions. Microbial-mineral surface interactions were determined using the direct observational technique, Fluid Tapping Mode™ Atomic Force Microscopy (TMAFM) in combination with measurements of ferrous iron concentrations and pH of the incubating solutions. Observations of live bacteria-surface interactions exposed to aerobic conditions showed localized pitting on Fe3+ oxyhydroxide coated quartz surfaces within 72 h of incubation. These pits corresponded directly to sites of bacterial surface adhesion and the extent of pitting was accompanied by the accumulation of ferrous iron to low but steady-state concentrations. Localized pitting was not observed on any Al3+ oxyhydroxide coated surfaces. In contrast, iron coated surfaces exposed to bacteria under anaerobic conditions revealed progressive, nonlocalized Fe loss over 96 h. This correlated with a temporal increase in ferrous iron concentrations in the bacteria-exposed solutions compared to the abiotic controls. Aqueous chemical measurements combined with the Fluid TMAFM observations indicate biologically-catalyzed iron reduction under both aerobic and anaerobic incubation. The pitting mechanism observed under aerobic conditions is proposed to result from a redox reaction at the bacteria-iron interface followed by the reoxidation of Fe2+ onto the surface. The evidence suggests that bacteria under anaerobic conditions maximize rates of dissimilatory reduction by remaining passively mobile on the surface. The weaker bacterial adhesion under anaerobic conditions enhances opportunities for bacteria-iron mineral surface contact. These findings may improve our understanding of relationships between the redox environment and bacterial mobility in the subsurface.

Microbiological and Geochemical Characterization of Microbial Fe(III) Reduction in Salt Marsh Sediments

Population densities of anaerobic Fe(III)-reducing bacteria (FeRB) and aerobic heterotrophs were inversely correlated in the surficial (0-2 cm) layers of Sapelo Island, Georgia, salt marsh sediments. In surficial sediments where densities of aerobic heterotrophs were low, the density of culturable FeRB correlated positively with the concentration of amorphous Fe(III) oxyhydroxides extractable by ascorbate. High FeRB densities and a decrease with depth of ascorbate-extractable Fe(III) were observed in the upper 6 cm of a tidal creek core. Culturable sulfate-reducing bacteria (SRB) and SRB-targeted rRNA signals were also detected in the upper 6-cm depth. The disappearance of FeRB below 6 cm, however, coincided with a large increase in the abundance of SRB. Thus, when FeRB are not limited by the availability of readily reducible amorphous Fe(III) oxyhydroxides, FeRB may outcompete SRB for growth substrates. Shewanella putrefaciens- and Geobacteraceae-targeted rRNA signals were at or below detection limits in all sediment samples, indicating that these FeRB are not predominant members of the active FeRB populations. The ubiquitous presence of FeRB at the sites studied challenges the traditional view that dissimilatory Fe(III) reduction is not an important pathway of organic carbon oxidation in salt marsh sediments.Population densities of anaerobic Fe(III)-reducing bacteria (FeRB) and aerobic heterotrophs were inversely correlated in the surficial (0-2 cm) layers of Sapelo Island, Georgia, salt marsh sediments. In surficial sediments where densities of aerobic heterotrophs were low, the density of culturable FeRB correlated positively with the concentration of amorphous Fe(III) oxyhydroxides extractable by ascorbate. High FeRB densities and a decrease with depth of ascorbate-extractable Fe(III) were observed in the upper 6 cm of a tidal creek core. Culturable sulfate-reducing bacteria (SRB) and SRB-targeted rRNA signals were also detected in the upper 6-cm depth. The disappearance of FeRB below 6 cm, however, coincided with a large increase in the abundance of SRB. Thus, when FeRB are not limited by the availability of readily reducible amorphous Fe(III) oxyhydroxides, FeRB may outcompete SRB for growth substrates. Shewanella putrefaciens- and Geobacteraceae-targeted rRNA signals were at or below detection limits in...

Sulfur-mediated electron shuttling during bacterial iron reduction

How bacteria manage to breathe on rust In the absence of oxygen, anaerobic bacteria turn to other chemical compounds during respiration. This can be helpful in detoxifying heavy-metal pollution. Flynn et al. (see the Perspective by Friedrich and Finster) found that alkaline conditions prevent a detoxifying bug—Shewanella oneidensis—from using enzymes to reduce rust-like minerals. Instead, the bacteria reduce elemental sulfur compounds, generating hydrogen sulfide that reduces the iron indirectly. This interplay between anoxic biogeochemical cycles may explain why some anaerobic bacteria contain the genetic machinery necessary to reduce multiple compounds besides oxygen. Science, this issue p. 1039; see also p. 974 Bacterial respiration of ferric iron involves sulfur intermediates in alkaline conditions [Also see Perspective by Friedrich and Finster] Microbial reduction of ferric iron [Fe(III)] is an important biogeochemical process in anoxic aquifers. Depending on groundwater pH, dissimilatory metal-reducing bacteria can also respire alternative electron acceptors to survive, including elemental sulfur (S0). To understand the interplay of Fe/S cycling under alkaline conditions, we combined thermodynamic geochemical modeling with bioreactor experiments using Shewanella oneidensis MR-1. Under these conditions, S. oneidensis can enzymatically reduce S0 but not goethite (α-FeOOH). The HS– produced subsequently reduces goethite abiotically. Because of the prevalence of alkaline conditions in many aquifers, Fe(III) reduction may thus proceed via S0-mediated electron-shuttling pathways.

Anaerobic Respiration of Elemental Sulfur and Thiosulfate by Shewanella oneidensis MR-1 Requires psrA, a Homolog of the phsA Gene of Salmonella enterica Serovar Typhimurium LT2

ABSTRACT Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of anaerobic terminal electron acceptors, including the inorganic sulfur compounds sulfite (SO32−), thiosulfate (S2O32−), tetrathionate (S4O62−), and elemental sulfur (S0). The molecular mechanism of anaerobic respiration of inorganic sulfur compounds by S. oneidensis, however, is poorly understood. In the present study, we identified a three-gene cluster in the S. oneidensis genome whose translated products displayed 59 to 73% amino acid similarity to the products of phsABC, a gene cluster required for S0 and S2O32− respiration by Salmonella enterica serovar Typhimurium LT2. Homologs of phsA (annotated as psrA) were identified in the genomes of Shewanella strains that reduce S0 and S2O32− yet were missing from the genomes of Shewanella strains unable to reduce these electron acceptors. A new suicide vector was constructed and used to generate a markerless, in-frame deletion of psrA, the gene encoding the putative thiosulfate reductase. The psrA deletion mutant (PSRA1) retained expression of downstream genes psrB and psrC but was unable to respire S0 or S2O32− as the terminal electron acceptor. Based on these results, we postulate that PsrA functions as the main subunit of the S. oneidensis S2O32− terminal reductase whose end products (sulfide [HS−] or SO32−) participate in an intraspecies sulfur cycle that drives S0 respiration.

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic-Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic-Fe(III) complexes to include Shewanella oneidensis MR-1. Soluble organic-Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O(2), nitrate or trimethylamine-N-oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic-Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)-citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic-Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild-type and Sol mutant strains, and all four Sol mutants retained wild-type siderophore production capability. Results of this study indicate that the production of soluble organic-Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.

Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1

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.

Design and application of a rapid screening technique for isolation of selenite reduction-deficient mutants of Shewanella putrefaciens

A rapid screening technique for isolation of selenite (Se(IV)) reduction-deficient (Ser) mutants was developed and used to identify four Ser mutants of Shewanella putrefaciens. Two Ser mutants were unable to grow anaerobically on fumarate, nitrate or nitrite. Two other Ser mutants were unable to grow anaerobically on all compounds tested as sole terminal electron acceptor. Previously isolated Mn(IV) reduction-deficient mutants displayed Ser-positive phenotypes and reduced Se(IV) at wild-type rates, while two of nine Fe(III) reduction-deficient mutants displayed Ser-negative phenotypes and reduced Se(IV) at low rates. This study provides the first reported method for isolation of Ser mutants and demonstrates that Se(IV) reduction by S. putrefaciens is respiratory chain-linked.