Plutonium(V/VI) Reduction by the Metal-Reducing Bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1.

We examined the ability of the metal-reducing bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1 to reduce Pu(VI) and Pu(V). Cell suspensions of both bacteria reduced oxidized Pu [a mixture of Pu(VI) and Pu(V)] to Pu(IV). The rate of plutonium reduction was similar to the rate of U(VI) reduction obtained under similar conditions for each bacteria. The rates of Pu(VI) and U(VI) reduction by cell suspensions of S. oneidensis were slightly higher than the rates observed with G. metallireducens. The reduced form of Pu was characterized as aggregates of nanoparticulates of Pu(IV). Transmission electron microscopy images of the solids obtained from the cultures after the reduction of Pu(VI) and Pu(V) by S. oneidensis show that the Pu precipitates have a crystalline structure. The nanoparticulates of Pu(IV) were precipitated on the surface of or within the cell walls of the bacteria. The production of Pu(III) was not observed, which indicates that Pu(IV) was the stable form of reduced Pu under these experimental conditions. Experiments examining the ability of these bacteria to use Pu(VI) as a terminal electron acceptor for growth were inconclusive. A slight increase in cell density was observed for both G. metallireducens and S. oneidensis when Pu(VI) was provided as the sole electron acceptor; however, Pu(VI) concentrations decreased similarly in both the experimental and control cultures.

Petrobactin is produced by both pathogenic and non-pathogenic isolates of the Bacillus cereus group of bacteria.

Petrobactin is the primary siderophore synthesized by Bacillus anthracis str Sterne and is required for virulence of this organism in a mouse model. The siderophore's biosynthetic machinery was recently defined and gene homologues of this operon exist in several other Bacillus strains known to be mammalian pathogens, but are absent in several known to be harmless such as B. subtilis and B. lichenformis. Thus, a common hypothesis regarding siderophore production in Bacillus species is that petrobactin production is exclusive to pathogenic isolates. In order to test this hypothesis, siderophores produced by 106 strains of an in-house library of the Bacillus cereus sensu lato group were isolated and identified using a MALDI-TOF-MS assay. Strains were selected from a previously defined phylogenetic tree of this group in order to include both known pathogens and innocuous strains. Petrobactin is produced by pathogenic strains and innocuous isolates alike, and thus is not itself indicative of virulence.

Effect of exogenous reductant on growth and iron mobilization from ferrihydrite by the Pseudomonas mendocina ymp strain.

Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations. This shows that exogenous reductants play a substantial role in the overall microbial iron bioavailability. The exogenous reductants may work together with the siderophores, Fe-scavenging agents, to facilitate ferrihydrite dissolution.

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS.

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D (1)H, (13)C and (15)N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 microM to 2.0 microM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation.

Enhanced exopolymer production and chromium stabilization in Pseudomonas putida unsaturated biofilms.

Chromium-contaminated soils threaten surface and groundwater quality at many industrial sites. In vadose zones, indigenous bacteria can reduce Cr(VI) to Cr(III), but the subsequent fate of Cr(III) and the roles of bacterial biofilms are relatively unknown. To investigate, we cultured Pseudomonas putida, a model organism for vadose zone bioremediation, as unsaturated biofilms on membranes overlaying iron-deficient solid media either containing molecular dichromate from potassium dichromate (Cr-only treatment) or with deposits of solid, dichromate-coated hematite (Fe+Cr treatment) to simulate vadose zone conditions. Controls included iron-deficient solid medium and an Fe-only treatment using solid hematite deposits. Under iron-deficient conditions, chromium exposure resulted in lower cell yield and lower amounts of cellular protein and carbohydrate, but providing iron in the form of hematite overcame these toxic effects of Cr. For the Cr and Fe+Cr treatments, Cr(VI) was completely reduced to Cr(III) that accumulated on biofilm cells and extracellular polymeric substances (EPSs). Chromium exposure resulted in elevated extracellular carbohydrates, protein, DNA, and EPS sugars that were relatively enriched in N-acetyl-glucosamine, rhamnose, glucose, and mannose. The proportions of EPS protein and carbohydrate relative to intracellular pools suggested Cr toxicity-mediated cell lysis as the origin. However, DNA accumulated extracellularly in amounts far greater than expected from cell lysis, and Cr was liberated when extracted EPS was treated with DNase. These results demonstrate that Cr accumulation in unsaturated biofilms occurs with enzymatic reduction of Cr(VI), cellular lysis, cellular association, and extracellular DNA binding of Cr(III), which altogether can facilitate localized biotic stabilization of Cr in contaminated vadose zones.

Actinide and metal toxicity to prospective bioremediation bacteria.

Bacteria may be beneficial for alleviating actinide contaminant migration through processes such as bioaccumulation or metal reduction. However, sites with radioactive contamination often contain multiple additional contaminants, including metals and organic chelators. Bacteria-based bioremediation requires that the microorganism functions in the presence of the target contaminant, as well as other contaminants. Here, we evaluate the toxicity of actinides, metals and chelators to two different bacteria proposed for use in radionuclide bioremediation, Deinococcus radiodurans and Pseudomonas putida, and the toxicity of Pu(VI) to Shewanella putrefaciens. Growth of D. radiodurans was inhibited at metal concentrations ranging from 1.8 microM Cd(II) to 32 mM Fe(III). Growth of P. putida was inhibited at metal concentrations ranging from 50 microM Ni(II) to 240 mM Fe(III). Actinides inhibited growth at mM concentrations: chelated Pu(IV), U(VI) and Np(V) inhibit D. radiodurans growth at 5.2, 2.5 and 2.1 mM respectively. Chelated U(VI) inhibits P. putida growth at 1.7 mM, while 3.6 mM chelated Pu(IV) inhibits growth only slightly. Pu(VI) inhibits S. putrefaciens growth at 6 mM. These results indicate that actinide toxicity is primarily chemical (not radiological), and that radiation resistance does not ensure radionuclide tolerance. This study also shows that Pu is less toxic than U and that actinides are less toxic than other types of metals, which suggests that actinide toxicity will not impede bioremediation using naturally occurring bacteria.

Petrobactin is the primary siderophore synthesized by Bacillus anthracis str. Sterne under conditions of iron starvation.

The siderophores of Bacillus anthracis are critical for the pathogen's proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of 1H and 13C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.