| 1. | A method for the reduction of an oxidized metal, comprising contacting the oxidized metal with a culture of a photosynthetic Proteobacterium or Rhodobacter in an aqueous medium under conditions facilitating reduction of the oxidized metal. |
| 2. | The method of claim 1 wherein conditions facilitating reduction of the oxidized metal comprise contacting with cellfree extracts or subcellular fractions from a culture or cultures of photosynthetic proteobacteria. |
| 3. | The method of claim 1 wherein the oxidized metal is a metal oxide or a metal oxyanion. |
| 4. | The method of claim 1 wherein the Proteobacterium is R. sphaeroides or R. capsulatus or JRhodoJ aσter sphaeroides 2.4.1, 2.4.7, 2.4.9, RS2, RS630, Si4, SWL, WS8 or J?. capsulatus BIO. |
| 5. | The method of claim 1 wherein the oxidized metal is reduced to a corresponding free metal. |
| 6. | The method of claim 1 wherein the oxidized metal is a metal oxide or oxyanion of selenium, tellurium or rhodium which is tellurate, tellurite, selenate, selenite or rhodium sesquioxide. |
| 7. | The method of claim 1 wherein conditions facilitating reduction of the metal oxide or oxyanion comprise an aerobic environment or anaerobic environment. |
| 8. | The method of claim 1 wherein conditions facilitating reduction of the oxidized metal comprise minimal medium having a carbon source in a low oxidation state such as a carboxylic acid or alcohol. |
| 9. | The method of claim 1 wherein conditions facilitating reduction of the metal oxide or oxyanion comprise autotrophic or heterotrophic conditions. |
| 10. | The method of claim 1 wherein heterotrophic conditions are photoheterotropic at an incident light intensity of between about 5 and 25 W/m2. |
| 11. | A method of removing oxidized metal from aqueous medium, comprising facilitating high level resistance of a photosynthetic Proteobacterium to the oxidized metal wherein the Proteobacterium is grown under aerobic or anaerobic conditions in minimal medium having a carbon source in a low oxidation state. |
| 12. | The method of claim 1 or claim 8 wherein the carbon source is butyrate, succinate, tartrate, acetate, ethanol, glycerol, a fatty acid, an alcohol or malate. |
| 13. | The method of claim 11 wherein the minimal medium contains about 0.2 to about 0.8% of the carbon source. |
| 14. | The method of claim 11 wherein the oxidized metal comprises silicon, molybdenum, arsenic, tungsten, tin, sulfur, antimony, europium or vanadium. |
| 15. | A microorganism having intrinsic resistance to tellurite characterized as a photosyntheticallycompetent strain of R. sphaeroides, 2.4.1ΔS, having the genotyp :Δ (42kb plasmid) . |
| 16. | A method of metal purification comprising the steps: growing R. sphaeroides in an aerobic or anaerobic environment in minimal medium; combining the R . sphaeroides with a sample containing a desired metal oxide or oxyanion; and isolating a cell fraction containing free metal. |
| 17. | The method of claim 16 wherein the J?. sphaeroides is 2.4.1, 2.4.7, 2.4.9 or 2.4.1ΔS. |
| 18. | The method of claim 16 wherein the cell fraction containing free metal is isolated from cytoplasmic membrane. |
| 19. | The method of claim 16 wherein the metal isolated is gold, platinum, uranium, silver, palladium, titanium, tellurium, selenium, europium or rhodium. |
| 20. | The method of claim 16 further comprising photoheterotrophic conditions. |
| 21. | A method of isolating substantially pure intra¬ cytoplasmic membrane, comprising growing R. sphaeroides in the presence of a heavy metal oxide or oxyanion under conditions facilitating reduction of the metal oxide or oxyanion and separating metalladen cytoplasmic membrane fractions from intracytoplasmic membrane to obtain an intracytoplasmic membrane fraction substantially free of contaminating cytoplasmic membrane. |
| 22. | A purified intracytoplasmic membrane prepared by the method of claim 22. |
| 23. | A method of hydrogen production, comprising culturing photosynthetic Proteobacterium or Rhodobacter in the presence of tellurite class oxyanions. |
| 24. | The method of claim 23 wherein the Rhodobacter is R. sphaeroides 2.4.1, 2.4.7, 2.4.9 or 2.4.1ΔS. |
| 25. | The method of claim 23 wherein the culturing is in the presence of carbon dioxide as a carbon source. |
| 26. | A method of converting carbon dioxide gas to biomass, comprising contacting the carbon dioxide gas with a culture of Proteobacterium or Rhodobacter in the presence of tellurite class oxyanions wherein the carbon dioxide is source of carbon. |
| 27. | The method of claim 23 or 26 wherein the culturing is in ambient or natural light. |
| 28. | The method of claim 23 or 26 wherein culturing includes gaseous nitrogen as nitrogen source. |
| 29. | The method of any of claims 1, 11, 16, 23 or 26 wherein the culturing is under heterotrophic or autotrophic conditions. |
| 30. | The method of any of claims 1, 11, 16, 23 or 26 wherein the culturing includes pyruvate as carbon source under anaerobic fermentation conditions. |
| 31. | The method of any of claims 1, 11, 16, 23 or 26 wherein culturing is anaerobic in the presence of carbon dioxide, nitrogen and hydrogen. |
BIOREDUCTION OF METAL OXIDES AND OXYANIONS
BY PHOTOBYNTHETIC BACTERIA
The United States Government may have certain rights in the present invention pursuant to Grant Number GM15590 and Grant Number GM31667 awarded by the National Institutes of Health. This is a continuation-in-part of U.S. patent application 07/820,116 filed January 13, 1992.
BACKGROUND OF THE INVENTION
Field of the Invention
The invention relates to microbiological methods of heavy-metal oxide or oxyanion removal from aqueous media by Rhodobacter sphaeroides . Several subgenera of _RήodoJaσter and related species efficiently reduce the metal oxides and oxyanions of selenium, tellurium, europium and rhodium to the free metal which is readily isolated from the cytoplasmic membrane. These microorganisms exhibit resistance to a wide variety of rare-earth oxides and oxyanions making bioremediation of selected heavy-metal oxides and oxyanions feasible, even in the presence of other oxides and/or oxyanions including those of vanadium, iodine, silicon, molybdenum, tin, tungsten, lead, reuthenium, antimony and arsenic.
Description of Related Art
A major environmental problem exists in dealing with toxic metal compounds found ubiquitously dispersed in groundwater, lakes, plant effluents, and aqueous waste. Generally these toxic compounds are heavy metal oxides or oxyanions exemplified by the tellurite, arsenate and periodate classes of rare-earth oxyanions and oxides. A
particularly obnoxious group of contaminants identified as a threat to western United States water supplies includes the oxyanions of selenium frequently found in agricultural wastewaters (Sylvester, 1988) .
Potential and actual health problems also arise due to toxic effects of many oxidized heavy metals. Exposure to tellurium compounds is hazardous to workers in the film and rubber industries, as well in battery manufacture. When accumulated in the human body, many of these elements have detrimental mental and physical effects (Schroeder et al . , 1967).
Bioremediation has been explored as a method of detoxification of toxic compounds found in water. Proposed methods generally take advantage of microbiological resistance to such compounds. The basis of resistance may be metabolic breakdown or concentration of the material within the microorganism. It is known, for example, that some species of Gram-positive bacteria, such as Corynebacterium diphtheriae, Streptococcus faecalis and most strains of Staphylococcus aureus are naturally resistant to tellurite and will often concentrate metallic tellurium inside the inner membrane (Walter and Taylor, 1989) . Resistance determinants to tellurite have been identified and isolated in Escherichia coli (Walter and Taylor, 1989) . However, resistance to tellurite is not a common property of bacteria and examples of naturally-occurring resistant strains are rare (Chiong et al . , 1988). Oftentimes such resistance is to only low or moderate levels of these compounds, e . g. < 100 μg/ l.
A method for accelerating recovery of selenium from aqueous streams is based on bioreduction of Se(VI) to Se(IV) with strains of the soil bacterium, Clostridium. A rapid exchange reaction between selenous acid and
pyrite is used to remove the selenium from solution. However, to remove selenium, further processing is required, e . g. , generation of hydrogen selenide and subsequent oxidization to the free metal (Khalafalla, 1990) . Clostridium species have also been utilized in a process for reducing waste- containing radionuclides or toxic metals, but the process requires obligate anaerobic conditions at elevated temperatures (Francis and Gillow, 1991) .
In addition to bioremediation, microorganisms are thought to have practical value in possible reclamation of metals from such sources as low grade ores, or in recovery processing. However, while a few bacterial species have resistance to one or more metal cations under some conditions, resistance may be based on accumulation rather than a metabolic reaction. Few microorganisms have been identified that reduce metal cations to the free metal (Summers and Silver, 1978) . Moreover, resistance may not be to whole classes of such compounds, but to only a few.
SUMMARY OF THE INVENTION
The present invention addresses one or more of the foregoing problems in providing a method to effectively bioreduce oxidized metals present in aqueous media. Under certain conditions, some members of the subgenera Rhodobacter sphaeroides will reduce oxyanions and oxides of selenium, tellurium, europium and rhodium to the free metal. The microorganisms survive in the presence of high levels of a wide variety of metal oxides and oxyanions, including those of arsenic, lead, tungsten, tin, sulfur, antimony, silicon and vanadium. The invention also includes a strain of Rhodobacter sphaeroides particularly effective in reducing oxyanions of tellurium to the free metal when the microorganism is
grown under photoheterotrophic conditions, allowing isolation of a cell fraction containing free metal. In this respect, efficient deposition of free metal occurs in the cytoplasmic membrane, but not the photosynthetic membrane. Rhodobacter sphaeroides will also accomplish the same reactions under chemoheterotrophic growth conditions.
The method of the present invention involves the use of the photosynthetic Proteobacteria. In one aspect of the invention the bacterium is useful for reducing oxidized metals in aqueous media. Metal oxides or metal oxyanions in a aqueous sample are contacted with particular members of the photosynthetic Proteobacteria which are grown under conditions that allow reduction of the oxidized metal. The species of Proteobacteria most useful in the practice of this invention are Rhodobacter sphaeroides or Rhodobacter capsulatus . Preferred strains include _R. sphaeroides 2.4.1, 2.4.7, 2.4.9, RS2, RS630, Si4, SWL, and WS8, and R. capsulatus BIO. Most preferred are strains 2.4.1 and 2.4.1ΔS of R. sphaeroides. There are other Proteobacteria (including members of the α-2 and α-3 phylogenetic subgroups) able to reduce metal oxides and metal oxyanions in aqueous solution. However, high-level resistance (HLR) to certain metal oxyanions has not been found in members of the ct-1, β-1 and γ-3 subgroups.
High level resistance of R. sphaeroides, particularly strain 2.4.1, has been shown with several classes of oxyanions, including the "tellurite class" of oxyanions. Typical oxyanions of this class include, for example, tellurate, tellurite, selenate, selenite, europium oxide and rhodium sesquioxide. Reduction of oxyanions in this class results in deposition of the pure metal, for example, metallic selenium, tellurium, rhodium
or europium in the cytoplasmic membrane of the microorganism employed.
Reduction of metal oxides or metal oxyanions from aqueous solutions in the presence of a bacterium such as R. sphaeroides is most preferably conducted under either aerobic or anaerobic conditions. Under aerobic conditions both R . sphaeroides and R . capsulatus express intrinsic HLR to tellurite with minimum inhibitory concentrations at least 80 times higher than minimum inhibitory concentrations previously described for E. coli , an enteric member of the 7-3 phylogenetic subgroup. Rhodopseudomonas palustris is an α-2 species. This microorganism expresses intrinsic resistance to tellurate that is 40 times greater than E. coli , while two photosynthetic members of the α-1 group and β-1 subgroups Rhodospirillum rubrum and Jϊ odocyσlus gelatinosus show low resistance to tellurite.
Generally, ~ intrinsic high-level resistance to metal oxides and/or metal oxyanions appears to occur in a number of species of purple non-sulphur bacteria during aerobic and anaerobic growth conditions. Moreover, the level of tellurite resistance appears to be strain- dependent. Minimum inhibitory concentration (MIC) for R. sphaeroides RS2 is approximately two- to three-fold lower than the MIC for either strain 2.4.1, 2.4.7, 2.4.9, Si4 or WS8. Generally, MICs are approximately 50% higher when cells are grown aerobically regardless of the strain or species, although one exception, R . gelatinosus has been found where there are no growth dependent differences in inhibitory metal oxyanion concentration, at least for tellurite.
Unexpectedly, composition of the medium in which the microorganism is grown has a significant effect on the resistance level of the microorganism to metal oxides or
metal oxyanions in aqueous solution. For example, rich media such as Luria-Bertani, yeast extract/peptone, or proteose-peptone medium are not conducive to high-level resistance. When grown in these media, R. sphaeroides 2.4.1 is sensitive to relatively low levels of the oxyanion. This is true whether or not the cultures are grown aerobically or anaerobically. A preferred medium is a minimal medium such as Sistro 's minimal medium A, ATCC medium 530, or Ormerod's photosynthetic minimal medium. High-level resistance to metal oxides or metal oxyanions decreases drastically when the medium is supplemented with peptone, casamino acids, tryptone or yeast extract. Surprisingly, there is strong evidence that inhibition of high-level resistance is due solely to the presence of a single amino acid, L-cysteine. Other compounds such as other amino acids or alternate electron acceptors such as trimethylamine-N-oxide or dimethyl sulfoxide (DMSO) do not appear to affect high-level resistance when added to minimal medium. With this knowledge, it is possible to eliminate the effect of L- cysteine and its inhibitory properties either by removing or destroying the L-cysteine, or by isolation of mutant strains unaffected by the L-cysteine.
Alternatively, and in addition to aerobic conditions in minimal medium, wild-type R. sphaeroides strains may be cultured anaerobically, either photosynthetically or employing anaerobic respiration. In preferred embodiments for the reduction of tellurite to tellurium metal, resistance in photosynthetically-grown cultures is directly proportional to incident light intensity. A preferred light intensity is 10W/m 2 which allows MICs at least two-fold higher than for cultures grown at 3W/m 2 . It is likely that optimal conditions in terms of light intensity should be developed for each metal desirous of being reduced. While 10W/m 2 has been found useful for the reduction of tellurite, other optimal light conditions
combined with appropriate culture medium may result in even higher MICs of tellurite as well as for other heavy-metal oxides and oxyanions. In practical terms, sunlight would probably be the preferred method of providing conditions conducive to encouraging high-level resistance in the photosynthetic bacteria.
JR odoJaσter sphaeroides 2.4.1 and other photosynthetic Proteobacteria may be grown by a variety of methods including aerobically (in shaking flasks, for example, or by sparging large liquid cultures with oxygen) ; anaerobically, either photosynthetically (in the presence of light using organic acids or carbon dioxide as a carbon source) or in the absence of light (with the addition of an alternate electron acceptor such as DMSO or TMAO to the growth medium) , or by fermentation of organic compounds such as pyruvate. They may also be grown photosynthetically in the presence of hydrogen and carbon dioxide. These organisms can also be grown employing nitrogen gas as the sole nitrogen source.
The invention also includes a means of facilitating high-level resistance of a photosynthetic Proteobacterium to metal oxides and oxyanions. Proteobacteria are grown aerobically in minimal medium preferably having a carbon source that has a low oxidation state. As used in this context, low oxidation state refers to carbon compounds that are highly reduced. A highly preferred carbon source is a dicarboxylic acid such as malate or succinate, or a monocarboxylic acid such as butyrate. There are numerous other low oxidation state carbon sources that may be used such as other organic acids and alcohols.
When grown in minimal medium having a carbon source in a low oxidation state, strains of R. sphaeroides exhibit high-level resistance to a wide variety of metal
oxides and oxyanions including silicon, molybendum, arsenic, tungsten, tin, sulphur, antimony, or vanadium. R. sphaeroides 2.4.1 in particular shows resistance to the oxides Moθ 3 , NH 4 V0 3 , Rh 2 0 3 «5H 2 0, Sb 2 0 3 , and Sn0 2 . Other oxyanions to which resistance was shown include I0 , Si0 3 2" , and Si0 2 ", as well as arsenate, molybdate, stannate, sulphite and tungstate.
A mutant photosynthetically competent strain of R. sphaeroides 2.4.1ΔS genotype is also part of the present invention. R. sphaeroides 2.4.lΔS is a derivative of R. sphaeroides 2.4.1 which has been "cured" of one of its five endogenous plas ids, the 42kb plasmid designed e (Fornari et al . , 1984) or "S" factor (Suwanto and Kaplan, 1989A; Suwanto and Kaplan, 1989B; Suwanto and Kaplan, 1991) . The plasmid is readily cured as described in Suwanto and Kaplan by the introduction of either of the incompatibility determinants, IncA or XπσB derived from native "S" factor on a selectable antibiotic resistance containing, unstable plasmid derivative. Once "S" is cured, the introduced plasmid is readily lost following removal of the antibiotic selection. Two important features of 2.4.1ΔS are that the phenotype associated with oxyanion or metal oxide metabolism is not associated with the "S" factor and that this strain may be used in conjugal genetic studies involving oriT mediated chromosome transfer.
Numerous genetic manipulations of R. sphaeroides are envisioned. Photosynthetic Proteobacteria may be genetically engineered to provide to the oxyanion and metal oxide metabolic properties associated with J?. sphaeroides 2.4.1. genotypes.
Yet another aspect of the present invention is metal purification utilizing R. sphaeroides. Generally, R. sphaeroides is cultured either under photoheterotrophic
or chemoheterotrophic growth conditions, generally described herein above. A sample containing a metal oxide or oxyanion is added, followed by isolation of a cell fraction containing the free metal.
Photoheterotrophic and chemoheterotrophic conditions for growing R. sphaeroides are described herein and may be varied somewhat depending on the particular strain of _R. sphaeroides employed. While the method is not limited to R . sphaeroides and may utilize any photosynthetic bacterium that shows high-level resistance, strains 2.4.1 and 2.4.1ΔS have demonstrated particularly high-level resistance to tellurite and are most preferred for reduction of tellurite to the free metal and subsequent isolation.
Metals isolated by the described method are typically localized in the cytoplasmic membrane. Isolation is readily accomplished by centrifugation and most preferably by sucrose density gradient centrifugation. The method works particularly well for metals such as selenium, tellurium, rhodium, europium and the like. The method is further contemplated to be useful for the isolation of such elements as gold, platinum, palladium, silver, titanium, iridium, germanium, plutonium, uranium, and the like, from their oxides, and oxyanion states.
Metal oxides or metal oxyanions reduced by this method appear to be located in a particular subcellular region, namely the cytoplasmic membrane. When shear forces are applied to such, as in a sucrose density gradient, the cytoplasmic membrane may be cleanly separated from other cellular constituents, including the photosynthetic membrane. This allows purification of the metal-laden cytoplasmic membrane and isolation of the free metal.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the mass spectrum of the headspace gas collected above photosynthetic (10 W/m 2 incident light intensity) cultures of R. sphaeroides grown either in the absence (Panel A) or presence (Panel B) of 250 μg/ml K 2 Te0 3 .
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention generally relates to Proteobacteria having the ability under certain conditions to efficiently reduce metal oxides and oxyanions to the free metal, and to their ability to survive in the presence of a wide range of metal oxides and oxyanions. The several examples following illustrate free metal deposition in a species of R . sphaeroides and its growth in the presence of toxic metal oxides and oxyanions. Selective growth conditions conducive to high level resistance of the microorganisms are also described.
The following examples are intended to illustrate the practice of the present invention and are not intended to be limiting. Numerous variations of growth conditions are envisioned which are expected to optimize for different metal oxides and oxyanions. It is also expected that one or more resistance factors, plasmid or chromosomal, identified with resistance will be isolated and sequenced, providing cassettes for transforming various host cells.
-Rhodobacter sphaeroides 2.4.1ΔS, ATCC Accession Number 49848 has been deposited with the American Type Culture Collection (ATCC) 12301 Parklawn Rd. , Rockville, MD 20852 under the Budapest Convention.
EXAMPLE 1
This example illustrates the intrinsic resistance of several species of Proteobacteria to tellurite. Intrinsic high-level resistance to tellurite is found in only a few species of these purple non-sulfur bacteria during chemoheterotrophic, anaerobic/dark, and photoheterotrophic growth conditions.
TeQ 3 2" Resistance in Proteobacteria
Several wild-type strains were grown either aerobically, anaerobically in the dark, or photoheterotrophically in minimal media in the presence of Te0 3 2" . Table 1 lists the bacterial strains tested for high level resistance to tellurite.
TABLE 1. Bacterial strains.
Organism/strain Relevant genotype/phenotype* Reference
Escherichia coli JM83 ara, A(lac-proAB), rpsL, thi, Messing, 1979 80dlacZ ΔM15
S17-1 C600: :RP-4, 2-Tc: :Mu: :Km: :Tn7 Simon, et al. , 1983 hsdR, hsdNC, recA
Rhodobacter capsulatus
BIO Wild-type Weaver, et al., 1975
Rhodobacter sphaeroides
TABLE 1. Bacterial strains.
Reference
Sockett, 1988
Neidle and Kaplan, 1992
Sistrom, 1977
Drews, 1966
Fornari and Kaplan, 1982
RS2 Wild-type Meinhardt, 1985
WS8 Wild-type, 1 endogenous plasmid Sistrom, 1977
Rhodocyclus gelatinosus str-1 Wild-type Uffen, 1976
Rhodopseudomonas palustris le5 Wild-type Firsow, 1977
Rhodopseudomonas viridis
F Wild-type Drews, 1966
Rhodospiήllum rubrum
Ha Wild-type
a Km r , Sp r , and Sm r denote resistance to kanamycin, spectinomycin, and streptomycin, respectively.
All Proteobacteria were grown at 30°C with the exception of E. coli which was cultured at 37°C on a Gyrotary shaker. Cultures of R . sphaeroides and R . gelatinosus were grown in LB, YP, or SMM containing either 0.4% succinate, 0.4% malate, or 0.4% butyrate as a carbon source. Cultures of R. capsulatus were grown in RCVB minimal medium containing 0.4% malate as a carbon source; R . rubrum was grown in SMM containing 0.4% malate, and 0.1% yeast extract. R. palustris and R.
viridis were grown in SMM containing 0.4% malate, 0.1% yeast extract, and 50 μg/ml each of p-aminobenzoic acid and cyanocobalamin. When necessary, antibiotics were added to growth media at the following final concentrations: kanamycin (Km), 25 μg/ml; spectinomycin (Sp) , 50 μg/ml; and streptomycin (Sm) , 50 μg/ml. Anaerobic-dark growth of J?. sphaeroides on SMM medium containing DMSO, and photoheterotrophic growth conditions have been previously reported.
Under aerobic conditions, both R. sphaeroides and R . capsulatus expressed intrinsic high level resistance to Te0 3 2* while virtually all other strains of bacteria tested showed much lower resistance under the same culture conditions. Results are shown in Table 2.
As indicated in Table 2, intrinsic high level resistance to tellurite occurred in only a few species of purple non-sulfur bacteria during aerobic and photoheterotrophic growth conditions. Moreover, the level of tellurite resistance was strain dependent: the MIC of K 2 Te0 3 for R. sphaeroides RS2 was approximately two-to three fold lower than the MIC for either strain 2.4.1, 2.4.7, 2.4.9, Si4, SWL or WS8. With the exception of R. gelatinosus, which exhibited no growth dependent difference in inhibitory Te0 3 2 " concentration, MICs were approximately 50% higher when cells were grown
"Based on the classification of Woese et al. b MICs were determined in the appropriate minimal synthetic medium at 30°C.
"Incident light intensity, 10 W/m 2 . d NA, not applicable.
aerobically, regardless of the strain or species. The JM83 and S17-1 strains of E. coli failed to grow in minimal medium containing 5 μg/ml K 2 Te0 3 .
EXAMPLE 2
The ability of R . sphaeroides to grow in the presence of selenium, tellurium, europium and rhodium oxyanions is demonstrated in the following example.
Growth of R. sphaeroides in the Presence of Te. Se, Eu or Rh-Containinq Oxyanions
Cells of R. sphaeroides 2.4.1 were grown in liquid medium as in Example 1. When medium contained Te0 3 or
Te0 4 2" , cells settled to the bottom of culture tubes over the course of the growth phase due to the intracellular accumulation of a dense metal deposit. Copious gas evolution was observed concomitant with cell growth. Centrifugation of broth cultures at 10000 x g resulted in a black cell pellet and a clear supernatant. Colonies of R. sphaeroides which formed on agar medium containing Te0 3 2" produced a black deposit which did not diffuse into the medium. Cells remained viable despite the accumulation of intracellular deposits: black colonies streaked onto agar medium containing no Te0 3 2* gave rise to normally pigmented colonies apparently through the dilution of metal complexes in the membranes of progeny cells.
Similar results were obtained for selenium, europium and rhodium containing compounds: when culture media contained Se0 3 2* or Se0 4 2" , the cells became bright red in color; in rhodium sesquioxide-containing media, the cells appeared grayish bronze. In europium oxide-containing media, the cells were grayish-white in appearance. The
relative toxicity of these five compounds to J? . sphaeroides was Se0 4 2 ">Te0 4 2 ">Te0 3 2" >Se0 3 2 ">Rh 2 0 3 »5H 2 0>Eu 2 0 3 .
EXAMPLE 3
The effect of culture conditions and medium composition on the high-level resistance of J?. sphaeroides to heavy-metal oxides was examined. Significant differences in resistance were found depending on the nature of the carbon source, incident light intensity and the presence of oxygen.
Effect of Culture Conditions and Medium Composition on High Level Resistance to TeQ 3 2"
R. sphaeroides 2.4.1 was grown either in complex or defined medium as indicated in Table 3.
While cultures of R . sphaeroides 2.4.1 grown in SMM expressed HLR to Te0 3 2 ", cells grown in rich media such as LB, YP, or proteose-peptone were sensitive to very low levels of the oxyanion, Table 3. This was true for cultures grown aerobically or anaerobically. Likewise, a thirty- to forty-fold reduction in Te0 3 2" resistance was observed when SMM was supplemented with either peptone,
Casamino acids, tryptone, or yeast extract. To determine if there was a single common component present in these supplements which was affecting HLR, SMM containing 0.4% succinate was supplemented individually with each of the twenty amino acids. This analysis indicated that a single amino acid, L-cysteine, was solely responsible for the increased sensitivity to Te0 3 2" . Neither cystine, glutathione nor thioglycollate, however, decreased HLR to Te0 3 2" when added to SMM, nor did the presence of alternate electron acceptors, such as trimethylamine-N- oxide or DMSO, Table 3. The fact that the addition of L- ethionine to SMM had no affect on HLR to Te0 3 2"
contrasted with previously studies with E. coli which demonstrated that exogenously supplied L-methionine enhanced Te0 3 2" resistance some two-fold (Scala and Williams, 1962; 1963). A similar inhibition of HLR by L- cysteine was also observed for Te0 4 2" , Se0 3 2" , and Se0 2" . Likewise, HLR to none of these compounds was enhanced by the addition of exogenous methionine.
TABLE 3. Effects of medium composition and growth conditions on HLR to TeO 3 2
MIC K 2 TeO 3 (μg/ml)
Medium 8 Supplement 13 Aerobic Photo¬ Ana synthetic erobic-
10 W/m 2 3 W/m 2 dark 41
Complex:
Luria-Bertani
Yeast Extract/Peptone
Proteose-peptone Defined: SMM+Butyrate SMM+Succinate SMM+ Malate SMM+Tartrate SMM+Glycerol SMM+Acetate SMM+Ethanol
SMM+Succinate 30 mM TMAO SMM+Succinate 1 mM L-Methionine SMM+Succinate 1 mM Cystine SMM+Succinate 1 mM Glutathione SMM+Succinate 1 mM Thioglycollate SMM+Succinate 0.3% Peptone SMM+Succinate 0.3% Yeast Extract SMM+Succinate 0.3% Tryptone SMM+Succinate 0.3% Casamino Acids SMM+Succinate 1 mM L-Cysteine a SMM contained 0.4% of the carbon source listed! b Supplement was added to culture medium to the final concentration listed. incident light intensity.
Supplemented with 60 mM DMSO.
Regardless of medium composition, the MIC of K 2 Teθ 3 for J?. sphaeroides 2.4.1 was always two- to three- old higher in aerobically- vs. photosynthetically-grown cultures. This was consistent with results obtained earlier for cells grown in succinate-containing SMM (Table 2) . Analyses also demonstrated that HLR to Te0 3 2" in photosynthetically-grown cultures was directly proportional to incident light intensity; in all the growth media examined, MICs were at least two-fold higher for cultures grown at 10 W/m 2 than for those grown at 3 W/m 2 , see Table 3.
A final observation with respect to medium composition concerned the effect the oxidation state of the carbon source had on the level of Te0 3 2* resistance in R. sphaeroides 2.4.1. While the MIC of K 2 Te0 3 for cells grown aerobically in SMM containing malate as the carbon source was 800 μg/ml, when more reduced carbon sources such as succinate or butyrate were substituted, the MICs increased to 900, and 1000 μg/ml, respectively. Similar results were also observed when cells were grown anaerobically in the light (photosynthetically) or anaerobically in the dark (in SMM containing DMSO) , see Table 3. These data suggested that the toxicity of Te0 3 2 " was inversely related to the oxidation state of the carbon source: the more reduced the carbon source, the higher the MIC of Te0 3 2_ .
EXAMPLE 4
This example illustrates the remarkable resistance of R. sphaeroides to a wide variety of rare earth oxides and oxyanions. The example is illustrated with strain 2.4.1 but similar resistance has been obtained with related strains such as Rhodobacter sphaeroides 2.4.1ΔS, 2.4.7, 2.4.9, Si4, SWL, WS8, RS2, RS630, 2.4.1-Ga;
_ odoJaσter capsulatus BIO; Rhodopseudomonas palustris le5; Rhodopseudomonas viridiε F.
A preferred strain used in some of the examples of the invention is Rhodobacter sphaeroides 2.4.1. This strain differs from the American Type Culture Collection strains (ATCC 17023, ATCC 11167, ATCC 14690, NCIB 8253 and NC1B827) which are also named as 2.4.1. The 2.4.1 strain used herein was originally provided by Dr. W.R. Sistrom over 20 years ago. It is believed that he received this strain from the laboratory of Dr. R.Y. Stanier, who in turn received it from Dr. C.B. Van Niel. It is unclear how the discrepancies in nomenclature between the 2.4.1 strain obtained from Dr. Sistrom and the ATCC strains arose.
The Rhodobacter sphaeroides 2.4.1 used herein may be obtained from Dr. Samuel Kaplan, The University of Health Science Center at Houston, Department of Microbiology and Molecular Genetics, P.O. Box 20708, Houston, Texas, USA 77225. An equally preferred strain is Rhodobacter sphaeroides 2.4.lΔs, which has ben deposited with the American Type Culture Collection, Rockville, MD 20852.
Additionally, other strains of photosynthetic proteobacteria exist which are likely to effect for metaloxide and oxyanion reduction, for example, other strains of R. sphaeroides commonly referred to as "2.4.1" (e.g., ATCC 11167, ATCC 14690, ATCC 17023, NCIB 8253, and NCIB 8287) that, while genetically distinct from 2.4.1 should be expected to carry out oxide and oxyanion reduction in a manner similar to 2.4.1.
Multiple-Oxyanion Hiσh-Level Resistance in R . sphaeroides
2.4.1
A total of twenty-seven rare-earth oxides and oxyanions were assayed for toxicity to R. sphaeroides 2.4.1. Results are shown in Table 4.
Twenty-seven rare-earth oxides and oxyanions, listed in Table 4, were assayed for toxicity to R. sphaeroides 2.4.1. Of those examined, only Cr0 3 , KRe0 4 , NaRu0 4 , K0s0 4 , Cr0 4 2" , and Mn0 4 " had MICs <20 μg/ml; the others had MICs in SMM >100 μg/ml under all growth conditions examined. Oxides having limited solubilities in SMM (e.g., Mo0 3 , PbO, Pb 2 θ 3 , Pb0 2 , Eu 2 0 3 , NH 4 V0 3 , Rh 2 0 3 »5H 2 0, Sb 2 0 3 , and Sn0 2 ) did not affect cell growth when present in growth media as saturated solutions. Only cultures grown in the presence of Te-, Se-, Eu or Rh-containing oxyanions evolved gas and accumulated intracellular deposits, Table 4. HLR to these six compounds was unaffected by extracellular P0 4 3 ", which suggested HLR to these compounds in R. sphaeroides 2.4.1 was not mediated by components of the phosphate-transport system. This would preclude any similarity between the mechanism of intrinsic HLR in R. sphaeroides and that encoded by the IncPα plasmid determinants, telA and telB (Walter et al . r 1991) .
a Photoheterotrophic growth in SMM containing succinate (10 W/m 2 incident light intensity. b Compounds with solubilities < 10 μg/ml did not inhibit growth in saturated solution.
"Medium contains 2 mM PO 4 3" , 10-fold lower than that of the standard formulation.
R. sphaeroides was also highly resistant to a second class of oxyanions, the "periodate class", but the resistance mechanism to this class differed significantly from that of the "tellurite class." Neither I0 4 " , Si0 3 2" , nor Si0 4 2* was reduced to its elemental state, and no gas evolution was observed. In sharp contrast to the "tellurite class," resistance to these oxyanions decreased three- to four-fold when the extracellular phosphate was reduced ten-fold. This suggested that resistance in R . sphaeroides 2.4.1 occurred as a result of reduced transport or increased efflux via a phosphate- transport system-mediated mechanism. It is interesting to note, however, that intrinsic resistance to these compounds in R. sphaeroides was still some twenty-fold greater than that of the 7-3 Proteobacteria (Summers and Silver, 1978).
A third class of oxyanions to which R. sphaeroides was highly resistant, the "arsenate class," was also examined. This group included arsenate, molybdate, stannate, sulfite, and tungstate. Similar to the "tellurite class" oxyanions, resistance to these compounds was unaffected by extracellular phosphate levels. In contrast, however, HLR to "arsenate class" compounds did not result in oxyanion reduction or intracellular metal sequestration. Like the "periodate class" oxyanions, these compounds were not reduced to their elemental states, and no gas was evolved. These data supported the existence of a third and distinctly different mechanism to effect HLR to "arsenate-class" oxyanions.
EXAMPLE 5
The ability of R . sphaeroides to concentrate tellurium metal in the cytoplasmic membrane is shown in
this example. The dense metal deposit was shown to be localized to the cytoplasmic membrane after a sucrose gradient isolation, leaving the intracytoplasmic (or photosynthetic) membrane unaffected.
Isolation of Tellurium from Membrane Fractions of R. sphaeroides 2.4.1
Two one-liter cultures of R . sphaeroides 2.4.1 were grown photoheterotrophically (10 W/m 2 incident light intensity) in SMM containing 0.4% succinate to a cell density of approximately 1.5 1.5 x 10 9 cells/ml. Prior to inoculation one flask was supplemented with 275 mg K 2 Te0 3 (equivalent to 138.3 mg Te ) to give a final medium concentration of 250 μg/ml K 2 Te0 3 . Following subcellular fractionation, the dense black deposit which accumulated within cells grown in Te0 3 2" -containing medium was localized to the cytoplasmic membrane via centrifugation through a discontinuous sucrose gradient. No metallic material was observed in the enriched chromatophore fraction (consisting of photosynthetic or intracytoplasmic membrane) at the 20:40% interface.
850 mg of crude membrane-metal complex was isolated. After purification and extraction with acetone:methanol and ethanol, 203 mg of a finely-divided metallic material resulted. Analysis of a 50.4 mg sample of this material identified 23.2 mg of Te° (a minimum net purity of 46%). The minimum Te° deposited in the one-liter culture, therefore, was 93 mg (203 mg x 0.46) or 0.7333 mmole.
Since the growth medium initially contained 138.3 mg of Te™, a minimum Te™ to Te° conversion of 67% was obtained.
Assay of the membrane fraction of cells grown in the absence of Te0 3 2" revealed no Te°, nor was any detected in the cytoplasmic or periplasmic fractions of either culture by this method. These results demonstrated
conclusively that J?. sphaeroides 2.4.1 could effect the intracellular reduction of Te™, which resulted in the deposition of metallic Te° in the cytoplasmic, but not intracytoplasmic, membrane.
Hydrogen Evolution From "Tellurite-Class" Oxyanion Reduction
Although no gas was evolved from cells grown in the absence of Te0 3 2" , 208 ml of water was displaced from the gas collection vessel over the culture grown in the presence of Te0 3 2" . This corresponded to 8.37 mole of gas (1 atm, 303°K), the major component of which was subsequently identified as H 2 by mass spectroscopy, Figure IB. While ionization products of H 2 0, N 2 , and C0 2 were detected in both samples, no H 2 was detected in the headspace over the control culture, Figure 1A. The trace amounts of Argon recorded in each spectra resulted from its use as a carrier in the analyses. Combined with earlier results, these data suggested approximately 11.5 mmoles of H 2 were evolved per mmole of Te° deposited.
EXAMPLE 6
The requirements for tellurite reduction in R. sphaeroides in vivo were determined by assaying tellurite resistance in a number of mutant strains.
Mechanism of TeQ 3 2" High Level Resistance in R. sphaeroides 2.4.1
Several growth conditions were examined to determine requirements for tellurite reduction in vivo. Table 5 indicates the growth conditions tested.
As indicated in Table 5, neither the DMSO reductase, the B800-850 spectral complex, nor the B875 spectral
complex was required to effect HLR to Te0 3 2 " under any growth condition examined. Deletion of the 42-kb endogenous plasmid of R . sphaeroides 2.4.1 did not diminish HLR to Te0 3 2" , although we did observe a 20% increase in Te0 3 2" sensitivity in the carotenoid-deficient strain, 2.4.l-Ga.
A single mutation in either prkB or cfxB diminished HLR to Te0 3 2' 10-20%, whereas strains deleted for either of their homologues, prkA or cfxA, were two-fold more sensitive to Te0 3 2" under aerobic growth conditions, and at least three- to five-fold more sensitive under photosynthetic and anaerobic-dark/DMSO growth conditions.
Analyses of additional J?. sphaeroides mutants determined the obligate requirement for an intact photosynthetic reaction center (RC) and a functional electron transport system for HLR to Te0 3 2 "when metabolic activities are carried out photosynthetically. These analyses also demonstrated that certain mutants, while unable to facilitate tellurite reduction, were resistant to intermediate concentrations of tellurite: viz a Bchl " mutant (MM1006) , a Puf * mutant (PUFB1) , and a strain deleted for cytochrome c 2 (CYCA1) were inhibited by 10 μg/ml K 2 Te0 3 under anaerobic-dark/DMSO growth conditions, but were unaffected by the addition of tellurite under aerobic growth. Likewise, the photosynthetically- incompetent double-deletion strains, CFXA * B" and PRKAT3 " , while unable to effect Te0 3 2" reduction either
TABLE 5. Analysis of intrinsic HLR to tellurite in R. sphaeroides mutants.
Relevant Photo¬ MIC K 2 TeO 3 (μg/ml) β genotype/ synthetic
Strain phenotype competence Aerobic Photosynthetic b Anaerobic-
10 W/m 2 dark 0
2.4.1 Wild-type 900 600 150
a MICs were determined in SMM containing succinate at 30°C. b Incident light intensity.
•Supplemented with 60 mM DMSO. d 0 indicates resistance to TeO 3 2" , but no deposition of Te°. e NG, no growth.
aerobically or anaerobically in the dark (in the presence of DMSO) , were resistant to tellurite at concentrations <200 μg/ml under aerobic conditions. In contrast, strains lacking either the RC-H polypeptide (PUHA1) or the cytochrome Jc j complex (BC17) were sensitive to 10 μg/ml K 2 Te0 3 under all growth conditions.
EXAMPLE 7
The experiments in this example were aimed at determining the intracellular localization of tellurite reductase activity.
Tellurite Reductase Activity in Cell Free Extracts
Subcellular fractions of aerobically grown cells were prepared from wild-type and three mutant strains unable to reduce tellurite. These cells were grown in the absence of Te0 3 2" , and were harvested during the mid- exponential phase of growth. This analysis, results of which are shown in Table 6, identified an FADH 2 -dependent Te0 3 2" reductase activity present in the membrane fraction of wild-type R . sphaeroides 2.4.1. Cells cultured in the presence of Te0 3 2" also expressed similar Te0 3 2' -dependent FADH 2 oxidation activity in vitro . A specific activity of 300 nmole FADH 2 /min per mg protein was detected in the membrane fraction of wild-type cells.
An FADH 2 -dependent Te0 3 2" reductase activity was also observed in the photosynthetically-incompetent strain
PRKA"B", despite this strain's inability to reduce Te0 3 2" in vivo, see Table 5. This suggested that in addition to an FADH 2 -dependent reductase, at least one other component was required to facilitate complete reduction to Te° in vivo.
Neither BC17 nor PUHA1, two mutants which were previously shown to be tellurite sensitive under both aerobic and anaerobic-dark/DMSO growth conditions, expressed significant levels of a Te0 3 2' -dependent FADH 2 oxidase activity in vitro . This may explain the inability of either to effect oxyanion reduction and metal sequestration in vivo .
Negligible reductase activity was observed in. the periplasmic and cytoplasmic fractions of all strains, and in separate analyses, a Te0 3 2" -dependent oxidation of NADH or NADPH was not detected in subcellular fractions from any of these strains. This would not preclude, however, the participation of a NADH- or NADPH-dependent oxidation step in the reduction of an intermediate in the reduction of Te™ to Te°.
EXAMPLE 8
This example illustrates the construction of a mutant R. sphaeroides from wild type strain 2.4.1.
R. sphaeroides 2. .AS
A mutant R. sphaeroides was prepared from wild type strain 2.4.1. J?. sphaeroides 2. . I Δs is a derivative of R . sphaeroides 2.4.1 which has been "cured" of one of its five endogenous plasmids, the 42-kb plasmid designated e (Fornari et al . , 1984) or "S" factor (Suwanto and Kaplan, 1989 A; Suwanto and Kaplan, 1989 B; Suwanto and Kaplan, 1991) .
The plasmid was readily cured by the introduction of either of the incompatibility determinants, IncA or IncB derived from native "S" factor on a selectable antibiotic resistance containing, unstable plasmid derivative. Once "S" was cured, the introduced plasmid was readily lost
following removal of the antibiotic selection. Two important features of 2.4.lΔS are that the phenotype associated with oxyanion or metal oxide metabolism is not associated with the "S" factor and this strain may be used in conjugal genetic studies involving oriT mediated chromosome transfer.
TABLE 6. Teθ 3 2 "-dependent FADH 2 oxidation in R. sphaeroides 2.4.1.
FADH oxidation"
Strain 8 Subcellular Fraction (nmole min" 1 mg" 1 )
2.4.1 Periplasm 1
Membrane 300
Cytoplasm 60
BC17 Periplasm 2
Membrane 51
Cytoplasm 20
PRKAΕ" Periplasm 3 Membrane 200 Cytoplasm 20
PUHA1 Periplasm 3
Membrane 28
Cytoplasm 37 a Cells were grown aerobically in SMM containing 0.4% succinate. b 100 μg/ml K 2 Te0 3 was used in all assays.
EXAMPLE 9
The ability of J?. sphaeroides to evolve molecular hydrogen and deposit metallic selenium in the cytoplasmic membrane under photoheterotrophic growth conditions is shown in this example.
Evolution of Hydrogen Gas from Cultures of R. sphaeroides 2.4.1 Grown Photoheterotrophically With Butyrate as a Carbon Source.
A ten-liter culture of R. sphaeroides 2.4.1 is grown photoheterotrophically at ambient temperatures of between 15 and 35°C. (using natural or artificial lighting having an intensity of between 1 and 200 W/m 2 ) in SMM containing 1000 μg/ml Na 2 Seθ 3 and up to 1% butyrate as a carbon source. The culture is maintained anaerobically by vigorous sparging with a mixture of 95% N 2 and 5% C0 2 , applied continuously throughout the course of growth. Mechanical mixing of the culture medium may be facilitated by an impeller assembly stirring at a rate of approximately 500 revolutions per minute, to increase yield.
A minimum of 85-105 mmole of hydrogen gas (1 atm, 303K) may be recovered from the headspace above the culture during the growth phase using an appropriate gas collection device. Concomitantly, cells may be harvested and lysed to recover the metallic selenium which has been deposited in the membrane fraction using the method according to Example 5.
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