APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2002, p. 3108–3113 Vol. 68, No. 60099-2240/02/$04.00�0 DOI: 10.1128/AEM.68.6.3108–3113.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Universal Immunoprobe for (Per)Chlorate-Reducing BacteriaSusan M. O’Connor and John D. Coates*

Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901

Received 26 December 2001/Accepted 1 April 2002

Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couplegrowth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identifiedtwo taxonomic groups, the Dechloromonas and the Dechlorosoma groups, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment. As part of these studies we demonstrated that chloritedismutation is a central step in the reductive pathway of (per)chlorate that is common to all ClRB and whichis mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highlyconserved among the ClRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an idealtarget for a probe specific for these organisms. Polyclonal antibodies were commercially raised against thepurified CD from the ClRB Dechloromonas agitata strain CKB. The obtained antiserum was deproteinated byammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of aserial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a highaffinity for the CD enzyme, and activity was observed in dilutions as low as 10�6 of the original antiserum. Theantiserum was active against both cell lysates and whole cells of D. agitata, but only if the cells were grownanaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to D.agitata, dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse ClRBrepresenting the alpha, beta, and gamma subclasses of Proteobacteria tested positive regardless of phylogeneticaffiliation. Interestingly, the dot blot response obtained for each of the ClRB cell lysates was different,suggesting that there may be some differences in the antigenic sites of the CD protein produced in theseorganisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related tothe ClRB which could not grow by (per)chlorate reduction. These studies have resulted in the development ofa highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in ClRB which canbe used to assess dissimilatory (per)chlorate-reducing populations in environmental samples regardless oftheir phylogenetic affiliations.

Environmental contamination with oxyanions of chlorine,especially perchlorate (ClO4

�), chlorate (ClO3�), chlorite

(ClO2�), and chlorine dioxide (ClO2), has been a constantly

growing problem over the last 100 years (33, 34). In generalthese compounds are not formed naturally, although one nat-ural source of perchlorate has been identified associated witha nitrate deposit in the Atacama desert region in northernChile (11, 17). These compounds have been introduced intothe environment in large quantities in the form of disinfec-tants, bleaching agents, and herbicides (3, 12, 29) and as com-ponents of explosives and rocket propellants by the aerospaceand defense industries (32). Legal discharge of perchlorate-containing wastestreams from munition manufacturing andhandling facilities prior to 1997 has recently been identified asthe predominant source of perchlorate found in major drinkingwater supplies in the United States (25, 34). Perchlorate con-tamination poses a significant health threat, as preliminarytoxicological studies have demonstrated that it has a directeffect on iodine uptake by the thyroid gland and at higherconcentrations (6 mg per kg of body weight per day) may resultin fatal bone marrow disease. As a result, the California En-vironmental Protection Agency initiated a recommended max-imum concentration limit of 18 �g/liter (25) which was laterincreased by the U.S. Environmental Protection Agency to 32

�g/liter (24). In 1998, perchlorate was added to the U.S. En-vironmental Protection Agency’s drinking water candidatecontaminant list.

The recent concerns over the environmental contaminationof groundwaters and drinking waters with perchlorate havefocused a significant amount of attention on microbial reme-diation (19, 24, 25, 33), as both chlorate and perchlorate can bereductively transformed into innocuous products by microor-ganisms in the absence of O2 (6, 9, 21, 26, 28, 30, 36). Thisreductive process was originally identified with chlorate (4) andwas associated with nitrate-respiring organisms which simplyused chlorate as a coincidental substrate for the nitrate reduc-tase (10, 13, 14). Growth was not associated with this metab-olism and chlorite was formed as a toxic end product (10, 13,14, 27). Now it is known that specialized organisms haveevolved which can grow by the anaerobic reductive dissimila-tion of (per)chlorate into innocuous chloride with simple or-ganic or inorganic electron donors (6, 7, 9, 18, 21, 22, 26, 28, 30,36). Recent studies have demonstrated that the ubiquity ofdissimilatory microbial (per)chlorate respiration is much moreextensive than was previously assumed (9). These studies re-sulted in the isolation and identification of more than 30 newdissimilatory (per)chlorate-reducing bacteria (ClRB) from abroad diversity of environments, including both pristine andcontaminated soils and sediments (9, 22; J. Pollock, L. A.Achenbach, and J. D. Coates, unpublished results). The knownClRB isolates represent a broad phylogeny with members inthe alpha, beta, gamma, and epsilon subclasses of Proteobac-

* Corresponding author. Mailing address: Department of Microbi-ology, Southern Illinois University, Carbondale, IL 62901. Phone:(618) 453-6132. Fax: (618) 453-8036. E-mail: jcoates@micro.siu.edu.

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teria (1, 9, 21); however, the majority of the known ClRB are inthe beta subclass of Proteobacteria (1, 9). 16S ribosomal DNA(rDNA) sequence analysis identified the Dechloromonas andDechlorosoma species as the two dominant ClRB genera in thebeta Proteobacteria (1), and several studies have demonstratedthat members of these two groups are present in nearly allenvironments screened, including both field samples and exsitu bioreactors treating perchlorate-contaminated wastes (9,20; Pollock et al., unpublished). Although pure culture studieshave demonstrated that members of these genera can growunder a broad range of environmental conditions, in generalthey grow optimally at circum-neutral pH in freshwater envi-ronments (6, 9, 22). In support of this, field studies have shownthat alternative ClRB are generally dominant in sites of ad-verse pH or salinity (Pollock et al., unpublished). As a result,it is difficult to rapidly determine which organisms are respon-sible for perchlorate removal in a given site. Although 16SrRNA primer sets have been designed which are specific forthe Dechloromonas and Dechlorosoma species (8; Pollock etal., unpublished), they are of limited use if these organisms arenot dominant in the contaminated environment. Due to theextreme phylogenetic diversity of ClRB, 16S rRNA primer setscan only be designed to detect a few specific genera in theenvironment; thus, there is a direct need for a more generalprobe that is specific to ClRB. This is especially true in light ofthe fact that several ClRB are essentially phylogenetically iden-tical, based on 16S rDNA sequence analysis, to non-(per)chlor-ate-reducing relatives; thus, even specific 16S rRNA primersets may prove unreliable, as they are not indicative of physi-ological functionality (2).

Previous studies (6, 9, 35) have demonstrated that chloritedismutation is a central step in the reductive pathway of(per)chlorate that is common to all ClRB and is mediated bythe enzyme chlorite dismutase (CD). If conserved, this enzymewould make an ideal target for a universal probe specific forthese organisms. We previously purified to homogeneity theCD from Dechloromonas agitata strain CKB (9). The purifiedCD was a homotetramer with a molecular mass of 120 kDa anda specific activity of 1,928 �mol of chlorite dismutated per mgof protein per min (9). This is similar to the molecular massand specific activity observed for the CD previously purifiedfrom the ClRB strain GR-1 (35). These data suggested thatsimilar CD enzymes can be found in the phylogenetically di-verse ClRB.

As part of these ongoing studies, polyclonal antibodies werecommercially raised against the purified CD from D. agitata todevelop an immunoprobe which would be universally selectivefor all ClRB regardless of their phylogenetic affiliation. Ourresults demonstrate the sensitivity, specificity, and applicabilityof this probe.

MATERIALS AND METHODS

Media and culturing techniques. All freshwater ClRB cultures, including D.agitata strain CKB, Dechloromonas aromatica strain RCB, Dechlorosoma suillumstrain PS, Pseudomonas sp. strain PK, and Dechlorospirillum anomolous strainWD were maintained on basal freshwater medium as previously described (6).The marine ClRB Dechloromarinus chlorophilus strain NSS was maintained onAPW medium as previously described (9). All ClRB were grown with acetate (10mM) as the electron donor and either chlorate or perchlorate (10 mM) as theelectron acceptor unless otherwise stated. In addition, active cultures of Pseudo-monas stutzeri, and Escherichia coli were grown aerobically on the same basal

freshwater medium as the ClRB from which the (per)chlorate was omitted.Rhodocyclus tenuis was grown on RCVB medium as previously described (31).

In the laboratory, standard anaerobic culturing techniques were used (5, 16,23). The anoxic basal medium was prepared by boiling under N2-CO2 (80:20[vol/vol]) to remove dissolved O2 and dispensed under N2-CO2 (80:20 [vol/vol])into anaerobic pressure tubes or serum bottles that were then capped with thickbutyl rubber stoppers and sterilized by autoclaving (15 min at 121°C).

Cell lysates were prepared from 1-liter cultures of the various organisms asfollows. The cells were harvested and resuspended in 5 ml of 10 mM phosphatebuffer, pH 7.3, and lysed by passing through a French press at 20,000 lb/in2. Thecell lysates were kept frozen at �80°C until required. The protein concentrationof both cell lysates and whole cells was determined using a Total Protein De-termination kit from Sigma (St. Louis, Mo.). The cell lysates and whole cells wereprobed with CD-specific immunoglobulin G (IgG) using the dot blot protocoloutlined below. The lysates and whole cells were diluted such that each spotcontained 5 �g of protein.

CD purification. The CD was purified from the soluble fraction of a lysed cellpreparation of D. agitata as previously described (9). Briefly, a 30-ml sample ofcell-free extract was loaded onto a 2.5- by 10-cm column packed with Q-Sepha-rose Fast Flow medium (Amersham Pharmacia Biotech, Piscataway, N.J.) andthe column was developed using a 0-to-300 mM KCl gradient in 50 mM Tris-HCl(pH 7.5). The CD active fractions were pooled and loaded onto a 2.5- by 20-cmcolumn packed with hydroxyapatite (Bio-Rad Laboratories, Hercules, Calif.) anddeveloped with a potassium phosphate buffer gradient (10 to 250 mM; pH 7.2).The resulting CD active fractions were pooled and augmented with ammoniumchloride to a final concentration of 2 M NH4Cl prior to loading onto a 1- by 1-cmphenyl-Sepharose high-performance column. The phenyl-Sepharose column wasdeveloped with a descending gradient of ammonium chloride (2 to 0 M) in 50mM Tris-HCl, pH 7.5. The eluted CD was concentrated by ultrafiltration (mo-lecular mass cutoff of 30 kDa) and passed through a 1.6- by 60-cm column packedwith Superdex 200 medium (Amersham Pharmacia Biotech). The pure CD waseluted with 150 mM NaCl in 50 mM potassium phosphate buffer (pH 7.2) andstored at �20°C until use.

CD-specific IgG. Polyclonal antibodies to purified CD were commerciallyraised by Capralogics Inc. (Hardwick, Mass.), following a standard operatingprocedure for the production of rabbit polyclonal antibodies. The antigen wasprepared by mixing CD with complete Freund’s adjuvant and immunizing NewZealand-type rabbits with 400 �g of the antigen by subcutaneous injection.Further boosts of 200 �g of antigen were provided every 3 weeks. The initial testbleed 4 weeks after the first immunization indicated the presence of CD-activepolyclonal antibodies. The rabbits were bled out after 12 weeks and the CDpolyclonal antibodies were partially purified from the antisera by ammoniumsulfate precipitation (15). One volume of a saturated solution of NH4SO4 wasadded to two volumes of antiserum to precipitate the polyclonal antibodies. Theresulting pellet was resuspended in a 0.25-volume of phosphate-buffered saline(PBS) and reprecipitated with one volume of saturated NH4SO4. The resultingIgG pellet was resuspended in a 0.5 volume of PBS and was dialyzed for 24 hagainst PBS. The protein concentration of the dialysate was determined by theBradford assay, and the purified CD polyclonal IgG was stored at �80°C in PBSat a final concentration of 15 mg/ml.

Dot blots. Dot blots of the CD-specific IgG were prepared by spotting strips ofImmobilon P transfer membranes (Millipore, Bedford, Mass.) with diluted pureCD (1 �l of a 5 �g/�l solution) and air dried. The membranes were then blockedfor 1 h in 0.1% Tween 20–PBS (TPBS) and incubated with the CD-specific IgGfor 1 h. The strips were washed four times in TPBS (5 min per wash) and werefurther incubated with goat anti-rabbit IgG (Sigma) labeled with horseradishperoxidase for 1 h in TPBS. The strips were washed as before and exposed to theSuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, Ill.) for 3min, after which the luminescence was recorded using Kodak BioMax film.

Western blotting. Pure CD and cell lysates from both D. agitata and E. coliwere run at 5 mA overnight on a sodium dodecyl sulfate (SDS)–12% polyacryl-amide gel electrophoresis (PAGE) gel prepared as previously described (9), afterwhich the gel was blotted onto Immobilon P transfer membrane, using Towbinbuffer (192 mM Glycine–25 mM Tris base–20% methanol, pH 8.3) and a semidrytransfer apparatus (Trans-Blot SD-Dry transfer cell; Bio-Rad) set at 20 V for 20to 30 min. The resulting blot transfer was developed as outlined above for the dotblot protocol, while the posttransfer gel was stained with Coomassie blue.

Fluorescence micrographs. Dry mounts of either D. agitata or E. coli cells wereprepared for fluorescence microscopy by spreading a small volume (approxi-mately 5 �l) of an active liquid culture onto an alcohol-cleaned glass slide. Theslides were air dried at 40°C and rinsed briefly in PBS (0.05 M; pH 7.2). Theprimary antibody (rabbit polyclonal) was then applied at a dilution of 1:50(diluted in PBS), and the slides were incubated for 30 min at room temperature

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after which the slide was again rinsed in PBS. The rinsed slides were then placedin a Coplin jar filled with PBS for an additional 10 min. After draining the slidesof excess PBS, the fluorescein-labeled secondary IgG (goat anti-rabbit) wasapplied at a dilution of 1:100 and allowed to stand for 30 min at room temper-ature. The slide was rinsed with PBS as above and a coverslip was placed on thesample with a mountant composed of 9 parts glycerol and 1 part PBS (pH 8.5).The prepared slides were examined in a Leica Orthoplan 2 epifluorescencemicroscope system, and images were recorded using conventional photographicprocedures or a Pixera 600CL Peltier-cooled charge-coupled device system.

RESULTS AND DISCUSSION

Sensitivity and specificity. The commercially raised anti-serum was partially purified by salt precipitation of serum

proteins using ammonium sulfate cuts prior to determinationof antigen binding activity. Dot blot analysis of a serial dilutionof the partially purified IgG against the purified CD (5 �g�l�1) indicated that the IgG had a high affinity for CD, andactivity was still observed at dilutions as low as 10�6 of theoriginal IgG (Fig. 1). No response was observed in similarexperiments performed with bovine serum albumin (5 �g�l�1) in place of the purified CD (data not shown). Westernblot analysis of cell lysates of D. agitata indicated that the IgGonly reacted with a single protein band in the cell lysate (Fig.2). No cross-reactivity of the IgG was observed with cell lysatesof E. coli (Fig. 2). The fact that the IgG reacted with thedenatured protein is indicative that the antigenic site of theIgG is located in the subunits and does not need the nativehomotetramer structure. Comparison of the reactive proteinband with the molecular mass marker indicated a molecularmass of 32 kDa (Fig. 2). This is similar to the migration of theCD previously observed in the cell lysates (9). Interestingly, thepurified CD migrated slightly further than the 32-kDa band ofthe lysed cell preparations (Fig. 2). In contrast, previous resultsof SDS-PAGE studies performed with freshly purified D. agi-tata CD demonstrated that its mobility was identical to the32-kDa band observed in the lysed cell preparation (9). Thedifference in the observed mobility is likely the result of theextended storage (18 months) of the purified CD at �20°Cprior to use, which may have resulted in a slight alteration ofthe protein structure and subsequent SDS-PAGE mobility pat-tern. In support of this hypothesis, activity determination of thestored CD indicated that the specific activity had decreased to569 �mol of chlorite dismutated per mg of protein per min,which is only 30% of the original activity (9).

Reaction with other ClRB. Microbial dissimilatory (per)chlorate reduction is a phylogenetically diverse metabolism,and microorganisms with this capability have been placed infour of the five subclasses of Proteobacteria (9). As such, theapplication of specific molecular probes based on signaturenucleotides in the 16S rDNA sequence are of limited use,especially in light of the fact that 16S rDNA sequence analysis

FIG. 1. Dot blot results of a range of dilutions of the CD-specificIgG. As shown, each sample analysis was performed in duplicate.

FIG. 2. SDS-PAGE gel and Western blot analysis of cell lysates from D. agitata and E. coli as well as the purified CD enzyme. Lanes 1 and 2,cell lysate from D. agitata; lanes 3 and 10, molecular mass marker; lanes 4 through 9, dilutions of purified CD; lane 11, cell lysate from E. coli.

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indicates that several ClRB are phylogenetically identical tonon-(per)chlorate-reducing organisms (2). To test the univer-sal applicability of the IgG, cell lysates of several phylogeneti-cally distinct ClRB representing the alpha, beta, and gammasubclasses of the Proteobacteria, including species of Dechlo-romonas, Dechlorosoma, Dechlorospirillum, Dechloromarinus,Ideonella, and Pseudomonas genera, were screened. In addi-tion, cell lysates of closely related organisms to the ClRB whichdo not grow by dissimilatory (per)chlorate reduction were alsoinvestigated. All the ClRB cell lysates tested positive regardlessof phylogenetic affiliation (Fig. 3). Interestingly, the responseobtained for each of the ClRB cell lysates was slightly different(based on dot blot intensity), suggesting that there may besome minor differences in the CD protein produced in theseorganisms (Fig. 3). Unsurprisingly, cell lysates of the Dechlo-romonas species, especially D. agitata, from which the CD waspurified, showed the most reactivity. No reaction was observedwith E. coli or with P. stutzeri, a phylogenetically identicalspecies of the ClRB Pseudomonas sp. strain PK based on 16SrDNA sequence analysis, which cannot grow by (per)chloratereduction (9) (Fig. 3). In contrast, a slight positive reaction wasobserved in the dot blot for the phototrophic beta proteobac-terium R. tenuis, which is the closest non-(per)chlorate-reduc-ing relative of the Dechloromonas genus (Fig. 3) (1, 6). How-

ever, if the dot blot procedure was repeated on this organismin the absence of the CD-specific IgG, the horseradish perox-idase-labeled goat anti-rabbit IgG, and the chemiluminescencesubstrate, a similar positive reaction was observed (Fig. 3). Thissuggests that the reaction was not a result of cross-reactivity ofthe IgG but was rather a reaction between the luminescentphototrophic centers of R. tenuis and the photographic filmused.

Reaction with whole cells. Interestingly, the IgG also reactedwith whole-cell preparations of the individual ClRB and notwith the negative controls (Fig. 3). In general, the reaction withthe whole cells was less intensive than that observed with thecell lysates. Previous observations made in our laboratory haveindicated that ClRB rapidly lyse in the absence of a suitableelectron donor or acceptor (8, 18). In order to determinewhether this reaction was the result of reaction of the IgG withCD released as a result of autolysis of the cells during the dotblot procedure, immunofluorescence micrographs were pre-pared from dry mounts of D. agitata by using fluorescein-labeled secondary goat anti-rabbit antibody. Phase-contrastmicroscopy of the dry mounts indicated that the cells of D.agitata were in good condition with little cell lysis (Fig. 4).Immunofluorescence microscopy revealed that the IgG readilyreacted with the whole cells and clearly obviated the intact cellstructure (Fig. 4). Similar studies performed with E. colishowed no fluorescence of the whole cells even after extendedstaining periods. The fact that the IgG reacted with the wholecells suggests that the CD may be present in the cell outermembrane, where it is accessible to the IgG. In support of this,previous studies demonstrated that the CD activity was asso-ciated with both the cell membrane and soluble fractions of alysed-cell preparation of D. agitata when prepared using aFrench press (6, 9). This result suggested that the CD wasloosely bound to the membrane and was sheared off by theFrench press procedure. In contrast, however, the CD activityof the ClRB strain GR-1 was located exclusively in the solublefraction (35).

Functional response. As the CD is a central enzyme in thepathway involved in the dissimilatory reduction of (per)chlor-ate, the CD-specific IgG may potentially be applied to deter-mining the metabolic state of ClRB. Dot blot analysis indicatedthat the CD-specific IgG was active only against cells of D.agitata grown with either perchlorate or chlorate as alternativeelectron acceptor (Fig. 5). No reaction was observed when D.agitata was grown aerobically (Fig. 5). Previous kinetics studiesby our lab on ClRB indicated that the enzymes involved inperchlorate reduction are induced only under anaerobic con-ditions in the presence of (per)chlorate (J. D. Coates, unpub-lished data). As part of these studies, we demonstrated that theCD activity was present only when the organisms were activelygrowing by (per)chlorate reduction. CD activity was not ob-served if the cells were growing aerobically or with nitrate as analternative electron acceptor. This implies that the CD-specificIgG will only bind to ClRB which are actively metabolizing(per)chlorate, as it is only under these conditions that theyproduce an active CD. We could not test the reactivity of theCD-specific IgG with nitrate-grown cells of D. agitata strainCKB, as this organism is one of the few ClRB that cannot growby nitrate reduction (6).

FIG. 3. Dot blot results of cell lysates and whole cells of a diverserange of ClRB and their non-(per)chlorate-reducing close relatives. Asshown, each sample analysis was performed in duplicate. �, organism isincapable of reductive (per)chlorate respiration.

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CONCLUSION

The present study resulted in the development of an immu-noprobe which has a high affinity for CD, a central enzymeinvolved in the dissimilatory reduction of (per)chlorate by

(per)chlorate-reducing bacteria. Previous studies indicatedthat this enzyme is unique to ClRB, and phylogenetically closerelatives of the known ClRB which cannot grow by reductionof (per)chlorate are incapable of the dismutation of chlorite (2,6, 9). In support of this, the developed immunoprobe wasspecific to ClRB and cross-reactivity with non-ClRB was notobserved, regardless of their phylogenetic similarities. Theprobe was reactive with both cell lysates and whole cells of allClRB tested but only when they were actively metabolizing(per)chlorate. Partiality based on phylogenetic affiliation wasnot observed, indicating that this probe has potential applica-tion for monitoring mixed (per)chlorate-reducing populationsin environmental samples.

ACKNOWLEDGMENTS

We thank John Bozzola of the IMAGE Center at Southern IllinoisUniversity for phase-contrast and immunofluorescence microscopy.

This work was supported by grant no. DACA72-00-C-0016 from theU.S. Department of Defense.

FIG. 4. Phase contrast and immunofluorescence micrographs of whole cells of D. agitata and E. coli stained with CD-specific IgG primaryantibody and fluorescein-labeled goat anti-rabbit IgG secondary antibody.

FIG. 5. Dot blot results of whole cells of D. agitata grown aerobi-cally or anaerobically with perchlorate as an electron acceptor. Asshown, each sample analysis was performed in duplicate.

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REFERENCES

1. Achenbach, L. A., R. A. Bruce, U. Michaelidou, and J. D. Coates. 2001.Dechloromonas agitata gen. nov., sp. nov. and Dechlorosoma suillum gen.nov., sp. nov., two novel environmentally dominant (per)chlorate-reducingbacteria and their phylogenetic position. Int. J. Syst. Evol. Microbiol. 51:527–533.

2. Achenbach, L. A., and J. D. Coates. 2000. Disparity between bacterial phy-logeny and physiology. ASM News 66:714–716.

3. Agaev, R., V. Danilov, V. Khachaturov, B. Kasymov, and B. Tishabaev. 1986.The toxicity to warm-blooded animals and fish of new defoliants based onsodium and magnesium chlorates. Uzb. Biol. Zh. 1:40–43.

4. Aslander, A. 1928. Experiments on the eradication of Canada thistle Cirsiumarvense with chlorates and other herbicides. J. Agric. Res. 36:915–935.

5. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979.Methanogens: reevaluation of a unique biological group. Microbiol. Rev.43:260–296.

6. Bruce, R. A., L. A. Achenbach, and J. D. Coates. 1999. Reduction of(per)chlorate by a novel organism isolated from a paper mill waste. Environ.Microbiol. 1:319–331.

7. Chaudhuri, S. K., J. G. Lack, and J. D. Coates. 2001. Biogenic magnetiteformation through anaerobic biooxidation of Fe(II). Appl. Environ. Micro-biol. 67:2844–2848.

8. Coates, J. D., R. A. Bruce, J. A. Patrick, and L. A. Achenbach. 1999. Hydro-carbon bioremediative potential of (per)chlorate-reducing bacteria. Bio-remed. J. 3:323–334.

9. Coates, J. D., U. Michaelidou, R. A. Bruce, S. M. O’Connor, J. N. Crespi, andL. A. Achenbach. 1999. The ubiquity and diversity of dissimilatory(per)chlorate-reducing bacteria. Appl. Environ. Microbiol. 65:5234–5241.

10. de Groot, G. N., and A. H. Stouthamer. 1969. Regulation of reductaseformation in Proteus mirabilis. I. Formation of reductases and enzymes of theformic hydrogenlyase complex in the wild type and in chlorate resistantmutants. Arch. Microbiol. 66:220–233.

11. Erickson, G. E. 1961. The Chilean nitrate deposits. Am. Sci. 71:366–374.12. Germgard, U., A. Teder, and D. Tormund. 1981. Chlorate formation during

chlorine dioxide bleaching of softwood kraft pulp. Paperi Puu 3:127–133.13. Hackenthal, E. 1965. Die Reduktion von Perchlorat durch Bacterien. II. Die

Identitat der Nitratreduktase und des Perchlorat Reduzierenden Enzyms ausB. cereus. Biochem. Pharm. 14:1313–1324.

14. Hackenthal, E., W. Mannheim, R. Hackenthal, and R. Becher. 1964. DieReduktion von Perchlorat durch Bakterien. I. Untersucungen an IntakenZellen. Biochem. Pharm. 13:195–206.

15. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

16. Hungate, R. E. 1969. A roll tube method for cultivation of strict anaerobes.Methods Microbiol. 3:B117–B132.

17. Konnert, J. A., H. T. J. Evans, Jr., J. J. McGee, and G. E. Ericksen. 1994.Mineralogical studies of the nitrate deposits of Chile: two new saline min-erals with the composition K6(Na, K)aNa6Mg10(XO4)12(IO)12.12(H2): fu-enzalidaite (X � S) and carlosruizite (X � Se). Am. Mineral. 79:1003–1008.

18. Lack, J. G., S. K. Chaudhuri, R. Chakraborty, L. A. Achenbach, and J. D.

Coates. Anaerobic biooxidation of Fe(II) by Dechlorosoma suillum. Microb.Ecol., in press.

19. Logan, B. E. 2001. Assessing the outlook for perchlorate remediation. En-viron. Sci. Technol 35:483A–487A.

20. Logan, B. E., H. Zhang, P. Mulvaney, M. G. Milner, I. M. Head, and R. F.Unz. 2001. Kinetics of perchlorate- and chlorate-respiring bacteria. Appl.Environ. Microbiol. 67:2499–2506.

21. Malmqvist, A., T. Welander, E. Moore, A. Ternstrom, G. Molin, and I.-M.Stenstrom. 1994. Ideonella dechloratans gen. nov., sp. nov., a new bacteriumcapable of growing anaerobically with chlorate as an electron acceptor. Syst.Appl. Microbiol. 17:58–64.

22. Michaelidou, U., L. A. Achenbach, and J. D. Coates. 2000. Isolation andcharacterization of two novel (per)chlorate-reducing bacteria from swinewaste lagoons, p. 271–283. In E. D. Urbansky (ed.), Perchlorate in theenvironment. Kluwer Academic/Plenum, New York, N.Y.

23. Miller, T. L., and M. J. Wolin. 1974. A serum bottle modification of theHungate technique for cultivating obligate anaerobes. Appl. Microbiol. 27:985–987.

24. Renner, R. 1999. EPA draft almost doubles safe dose of perchlorate in water.Environ. Sci. Technol. 33:110A–111A.

25. Renner, R. 1998. Perchlorate-tainted wells spur government action. Environ.Sci. Technol. 9:210A.

26. Rikken, G., A. Kroon, and C. van Ginkel. 1996. Transformation of(per)chlorate into chloride by a newly isolated bacterium: reduction anddismutation. Appl. Microbiol. Biotechnol. 45:420–426.

27. Roldan, M. D., F. Reyes, C. Moreno-Vivian, and F. Castillo. 1994. Chlorateand nitrate reduction in the phototrophic bacteria Rhodobacter capsulatusand Rhodobacter sphaeroides. Curr. Microbiol. 29:241–245.

28. Romanenko, V. I., V. N. Korenkov, and S. I. Kuznetsov. 1976. Bacterialdecomposition of ammonium perchlorate. Mikrobiologiya 45:204–209.

29. Rosemarin, A., K. Lehtinen, and M. Notini. 1990. Effects of treated anduntreated softwood pulp mill effluents on Baltic sea algae and invertebratesin model ecosystems. Nord. Pulp Paper Res. J. 2:83–87.

30. Stepanyuk, V., G. Smirnova, T. Klyushnikova, N. Kanyuk, L. Panchenko, T.Nogina, and V. Prima. 1992. New species of the Acinetobacter genus Acin-etobacter thermotoleranticus sp. nov. Mikrobiologiya 61:347–356.

31. Tayeh, M. A., and M. T. Madigan. 1987. Malate dehydrogenase in phototro-phic purple bacteria: purification, molecular weight, and quaternary struc-ture. J. Bacteriol. 169:4196–4202.

32. Urbanski, T. 1984. Chemistry and technology of explosives, vol. 4, p. 602–620. Pergamon Press, Oxford, United Kingdom.

33. Urbansky, E. T. 1998. Perchlorate chemistry: implications for analysis andremediation. Bioremed. J. 2:81–95.

34. U.S. Environmental Protection Agency. 2000. The effects of ammoniumperchlorate on thyroids. Pathology Working Group Report. [Online.] http://www.epa.gov/ncea/perch.htm.

35. van Ginkel, C., G. Rikken, A. Kroon, and S. Kengen. 1996. Purification andcharacterization of chlorite dismutase: a novel oxygen-generating enzyme.Arch. Microbiol. 166:321–326.

36. Wallace, W., T. Ward, A. Breen, and H. Attaway. 1996. Identification of ananaerobic bacterium which reduces perchlorate and chlorate as Wolinellasuccinogenes. J. Ind. Microbiol. 16:68–72.

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