Hydroxylation and Carboxylation--Two Crucial Steps of Anaerobic Benzene Degradation by Dechloromonas Strain RCB

Benzene is a highly toxic industrial compound that is essentialto the production of various chemicals, drugs, and fuel oils.Due to its toxicity and carcinogenicity, much recent attentionhas been focused on benzene biodegradation, especially in theabsence of molecular oxygen. However, the mechanism by whichanaerobic benzene biodegradation occurs is still unclear. Thisis because until the recent isolation of Dechloromonas strainsJJ and RCB no organism that anaerobically degraded benzene wasavailable with which to elucidate the pathway. Although manymicroorganisms use an initial fumarate addition reaction forhydrocarbon biodegradation, the large activation energy requiredargues against this mechanism for benzene. Other possible mechanismsinclude hydroxylation, carboxylation, biomethylation, or reductionof the benzene ring, but previous studies performed with undefinedbenzene-degrading cultures were unable to clearly distinguishwhich, if any, of these alternatives is used. Here we demonstratethat anaerobic nitrate-dependent benzene degradation by Dechloromonasstrain RCB involves an initial hydroxylation, subsequent carboxylation,and loss of the hydroxyl group to form benzoate. These studiesprovide the first pure-culture evidence of the pathway of anaerobicbenzene degradation. The outcome of these studies also suggeststhat all anaerobic benzene-degrading microorganisms, regardlessof their terminal electron acceptor, may use this pathway.

INTRODUCTION

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Introduction

Benzene has a broad range of industrial uses and is one of thetop 20 production volume chemicals in the United States, withan annual production of almost 9 million metric tons (16). Inaddition to its presence in petroleum-based fuels, benzene isoften used as a raw material for the manufacture of other chemicals,rubbers, lubricants, dyes, detergents, drugs, and pesticides.Other sources, including volcanoes, forest fires, and cigarettesmoke, also contribute to the benzene in the environment. Benzeneis among the most prevalent organic contaminants in groundwaterand is a major concern due to its toxicity and relatively highsolubility (16). It is currently ranked sixth on the U.S. NationalPriorities List, and has been found in at least 813 of the 1,430current or former National Priorities List sites (http://www.atsdr.cdc.gov/cxcx3.html).Benzene is biodegradable (16, 26), especially in the presenceof oxygen; however, when soils and sediments are contaminatedwith benzene, extensive anaerobic zones frequently develop dueto stimulation of the indigenous aerobic microbial population,resulting in rapid depletion of the dissolved oxygen contentof the groundwater (2, 11, 12, 27). As a result, a lot of attentionhas been focused on anaerobic benzene degradation over the pasttwo decades, and anaerobic benzene degradation has been demonstratedunder nitrate-reducing (9), Fe(III)-reducing (29, 30), sulfate-reducing(14, 18, 28), and methanogenic (21, 37) conditions. However,until recently, no organism existed in pure culture that wascapable of this metabolism (16, 26). Microbial community analysisof benzene-degrading sediments and enrichment cultures usingmolecular techniques has suggested that a functional role inanaerobic benzene degradation is played by members of the familyGeobacteraceae under Fe(III)-reducing conditions (33) and bymembers of the family Desulfobacteriaceae under both sulfate-reducingand methanogenic conditions (31, 35). However, there is no directevidence which shows that members of either of these familiesof organisms are capable of benzene degradation. Recently, Dechloromonasstrains RCB and JJ, the first two organisms capable of anaerobicbenzene degradation, were isolated from completely differentenvironments and described (15). Strains RCB and JJ belong tothe beta subclass of the Proteobacteria, exhibit 98.1% 16S rRNAgene sequence similarity, and metabolize benzene aerobicallyor anaerobically with nitrate as the sole electron acceptor(15). Anaerobically, Dechloromonas strain RCB completely degradesbenzene to CO₂, and concentrations as high as 160 µM areremoved within 5 days (15).

The biochemical pathway of anaerobic benzene degradation byany organism is currently unknown, but several possibilitiesexist (16). These include initial carboxylation, hydroxylation,and methylation with subsequent transformation to the centralaromatic intermediate benzoate or to its coenzyme A (CoA) thioesterform, benzoyl-CoA (16). This compound then undergoes furtherenzymatic ring reduction and ring cleavage, which forms 3-acetyl-CoAand CO₂. Previous studies of methanogenic benzene-degradingenrichments have indicated that phenol, cyclohexanone, propionate,and acetate are possible metabolites (21, 36, 37). Furthermore,phenol and benzoate were both detected as intermediates of benzenedegradation with sulfate-reducing and Fe(III)-reducing enrichments(10, 32). However, all of these studies were performed withundefined sediments or enrichment cultures, and whether thecompounds were formed directly from benzene catabolism by asingle organism or were formed as a result of several sequentialmetabolic steps involving several different organisms is unknown.

As part of our ongoing studies on the anaerobic degradationof benzene by Dechloromonas strain RCB, we investigated themetabolic intermediates formed with nitrate as the electronacceptor. The results of these studies provide the first documentationof the mechanism of anaerobic nitrate-dependent metabolism ofbenzene in pure culture and also provide significant insightinto the initial process of oxidation of this compound in theabsence of oxygen.

MATERIALS AND METHODS

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Materials and Methods

Media and stock preparation.
All media and solutions were prepared using strictly anaerobictechniques, as previously described (8, 15). All anaerobic culturingwas performed in sealed serum bottles under an N₂-CO₂ (80:20,vol/vol) headspace. Unless otherwise stated, all chemicals wereacquired from Sigma Chemicals, Missouri. Anoxic aqueous stocksolutions of propyl iodide (100 mM), sodium iodide (100 mM),5,5-dimethyl-1-pyrroline-N-oxide (1 M), mannitol (1 M), sodiumdithionite (100 mM), sodium nitrate (1 M), phenol (10 mM), benzoicacid (10 mM), and benzene (3.5 mM) were prepared under an N₂headspace. Amendments were made anaerobically as needed intoexperimental bottles from these anoxic stock solutions. StrainRCB was routinely cultured on anoxic minimal media (8) using10 mM acetate and 16 mM nitrate as the electron donor and acceptor,respectively, in 1-liter bottles sealed with thick butyl rubberstoppers under an N₂-CO₂ (80:20, vol/vol) headspace. Small amountsof benzene (10 µM) were added to the culture media duringgrowth and incubation of the cells to ensure induction of theappropriate biochemical pathway.

Sample preparation for analysis of benzene and its metabolites.
Samples (15 to 20 ml) of culture broth for analysis of volatilemetabolites of benzene were collected at regular time intervalsfrom active benzene-degrading cultures and extracted with diethylether (5 ml) by vigorous shaking for 90 s. The ether layer wasthen collected in Supelco screw-top clear vials with polytetrafluoroethylene/Neoprene-linedcaps and dried over anhydrous sodium sulfate to remove any tracesof water prior to injection using a Hamilton gas-tight syringeinto a gas chromatograph-mass spectrometer (GC-MS) for analysis.

Samples (25 to 30 ml) of culture broth for analysis of water-solublenonvolatile metabolites were first alkalized using 1 M NaOHto pH 12 for 30 min to cleave thioesters. This was followedby acidification of the samples for 30 min using concentratedHCl to approximately pH 2. Acidified samples were then extractedtwice using 10 to 15 ml ethyl acetate each time. The ethyl acetateextracts were combined and filtered over anhydrous sodium sulfateprior to concentration to approximately 1 ml by rotary evaporationat 80°C. The concentrated ethyl acetate extracts were subsequentlyevaporated to dryness under a steady stream of nitrogen gasand then redissolved using 0.5 ml of ethyl acetate before derivatizationwith 0.1 ml/sample of N,O-bis[trimethylsilyl]trifluoroacetamideand 1% trimethylchlorosilane (Pierce Chemicals, Rockford, Ill.)according to the manufacturer's instructions.

Analytical methods.
¹⁴CO₂ production in the headspace of active cultures amendedwith [¹⁴C]acetate (1 µCi) or [¹⁴C]benzene (1 µCi)was determined with a gas chromatograph equipped with gas proportionalcounting detection, as previously described (15). Both [¹⁴C]acetate(43 mCi/mmol) and [UL-¹⁴C]benzene (55mCi/mmol) were purchasedfrom Sigma.

Chemicals.
Analysis of ether extracts for benzene and phenol detectionwas performed using a gas chromatograph equipped with a massspectrometry detector (Shimadzu GCMS-QP2010) with electron ionization.The injector port temperature was set at 110°C with a 1:20split ratio, and the carrier gas used was helium flowing throughthe column at a rate of 1.48 ml · min^–1. Chromatographyof each sample was performed using an XTI-5 column (Restek Corp.,Pennsylvania). The column temperature was initially set at 40°Cfor 1 min and was subsequently ramped at a rate of 4°C min^–1to a final temperature of 125°C.

Analysis of aqueous-phase extracts for metabolite detectionwas performed similarly by GC-MS of 3-µl sample extracts.The injection port temperature was set at 110°C with a 1:50split ratio, and helium was used as the carrier gas at a flowrate of 1.46 ml · min^–1. Chromatography of eachsample was performed using an XTI-5 column which was temperatureramped at a rate of 4°C · min^–1 to a finaltemperature of 230°C after an initial 2-min hold at 80°C.The hold time at the final temperature was 5 min. In all cases,mass spectra data were characterized by comparison with thestandard National Institute of Standards and Technology (NIST)library, and compound identification was confirmed by comparisonwith chemical standards freshly prepared and derivatized usingthe sample preparation protocols outlined above.

Iodide determinations were performed by ion chromatography byusing a DX 500 ion chromatograph with suppressed conductivitydetection and an IonPac AS16 anion-exchange column (Dionex Corp.,Sunnyvale, CA). A mobile phase of 35 mM NaOH was used at a flowrate of 1 ml · min^–1.

Optical density at 600 nm was determined using a Cary 50 BioUV-visible spectrophotometer manufactured by Varian AnalyticalInstruments, Palo Alto, CA.

RESULTS AND DISCUSSION

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Results and Discussion

Phenol as a metabolite of benzene degradation.
GC-MS of ether extracts of an active benzene-degrading anaerobicculture revealed the presence of a single peak in addition tobenzene with a mass spectrum identical to that of phenol (datanot shown). The peak identification was confirmed by comparisonof mass spectra data with data from the NIST library and freshlyprepared chemical standards. The phenol peak appeared in samplescollected shortly after the onset of benzene degradation amendedwith 5 mM nitrate as the electron acceptor (Fig. 1). No benzenedegradation or phenol formation was observed in the absenceof the electron acceptor. The phenol concentration peaked atalmost 2 µM within 30 h of inoculation, at which pointalmost 19 µM benzene had been utilized by the active culture(Fig. 1). Once benzene was completely depleted, the phenol concentrationrapidly dropped to levels below detection (Fig. 1). When theculture was refed with 35 µM benzene, rapid benzene degradationwith concomitant phenol formation was again observed (Fig. 1).

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FIG. 1. Transient phenol formation resulting from anaerobic benzene degradation by strain RCB using nitrate as the sole electron acceptor. The data are averages of duplicate determinations.

Benzoate as a metabolite of benzene degradation.
In order to investigate the production of nonvolatile water-solubleintermediates formed during anaerobic nitrate-dependent benzenecatabolism by strain RCB, samples were collected and extractedwith ethyl acetate prior to derivatization by silylation forchromatographic analysis. GC-MS analysis revealed the presenceof a monoaromatic compound which was identified as benzoateby comparison of the mass spectra with data from the NIST libraryand authentic benzoate standards (data not shown). Benzoateaccumulated gradually to a concentration of almost 1.5 µMafter 70 h of incubation (Fig. 2). As observed with phenol,benzoate was formed concomitant with benzene degradation. Thetransient nature of benzoate observed correlated well with thetransient formation and removal of phenol (Fig. 2). Benzoateformation was not observed in cultures that were not amendedwith the electron acceptor nitrate.

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FIG. 2. Transient formation of benzoate and phenol during anaerobic degradation of benzene by active cultures of strain RCB using nitrate as the sole electron acceptor. The data are averages of duplicate determinations.

Sequence of catabolite production.
To determine the sequence of phenol and benzoate formation duringbenzene catabolism by strain RCB, anaerobic active cultureswere grown with 45 µM phenol as the sole electron donorand 5 mM nitrate as the electron acceptor. At regular intervalsthroughout the incubation, samples were collected and analyzedby GC-MS. Analysis revealed that benzoate was transiently formedby strain RCB concomitant with phenol degradation (Fig. 3).No benzoate was formed in cultures that were not amended witheither phenol or nitrate. After 24 h of incubation, almost 1.5µM benzoate was detected in the culture broth.

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FIG. 3. Transient formation of benzoate during anaerobic degradation of phenol by active cultures of strain RCB using nitrate as the sole electron acceptor. The data are averages of duplicate determinations.

In contrast, no phenol formation occurred in similar experimentsperformed with active cultures of strain RCB growing anaerobicallyon 50 µM benzoate with nitrate as the electron acceptor.These results indicate that phenol formation precedes formationof benzoate as an intermediate of anaerobic nitrate-dependentbenzene oxidation by strain RCB.

Location of intermediate formation.
In order to determine if the intermediates were formed extracellularlyor were extracted from the intracellular cytoplasmic pools ofwhole cells of strain RCB during sample preparation, subsamplesfrom active benzene-oxidizing culture broths were prefilteredthrough 0.2-µm-pore-size filters to remove the cells priorto extraction and analysis. GC-MS analysis indicated that thephenol levels in extracts of filtered and unfiltered culturebroths were identical throughout the incubation (Fig. 4), suggestingthat the hydroxylation reaction occurs on the outer membraneor in the periplasm of the cell, from which the phenol readilydiffuses into the culture milieu. Like the phenol levels, thebenzoate levels were identical in extracts of both the filtered(0.2-µm-pore-size filters) and unfiltered culture brothsthroughout the incubation (data not shown), implying that benzoateformation also occurs on the outer membrane or in the periplasmof strain RCB.

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FIG. 4. Concentrations of phenol and benzoate in samples prepared from filtered and unfiltered culture broths of an active benzene-degrading culture of strain RCB. The data are averages of duplicate determinations.

Role of molecular oxygen.
Hydroxylation of benzene to phenol is the accepted primary routeof benzene metabolism by mammalian systems (25). However, thisreaction is catalyzed by a monooxygenase P450 cytochrome isozymeand is dependent on the availability of molecular oxygen. Toensure that the benzene degradation in the active cultures ofstrain RCB was not due to a similar mechanism involving molecularoxygen, experiments were performed in anoxic culture media amendedwith 0.5 mM sodium dithionite or 0.1 mM sodium ascorbate asan alternative chemical reductant to remove any traces of dissolvedO₂ (Fig. 5). In spite of the presence of the chemical reductantsat the concentrations tested, [¹⁴C]benzene was rapidly oxidizedto¹⁴CO₂ in these experiments only in the presence of nitrate(5 mM) (Fig. 5), discounting any possible role for molecularoxygen in the metabolism. GC-MS analysis revealed the formationof phenol concomitant with benzene degradation in culture brothsamended with sodium dithionite (data not shown). In addition,no phenol was detected in benzene-degrading cultures of strainRCB incubated with oxygen as the electron acceptor (data notshown). This result is consistent with previous reports of studieson the metabolites of aerobic microbial benzene degradation,in which phenol was not detected as an intermediate (19).

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FIG. 5. Effect of the presence and absence of chemical reducing agents on [¹⁴C]benzene oxidation by active benzene-degrading cultures of strain RCB with nitrate as the electron acceptor. The data are averages of triplicate determinations.

It is interesting that although the metabolism was not inhibited,addition of dithionite as a reducing agent (0.5 mM) retardedthe rate of benzene degradation and the subsequent phenol formationcompared to unamended controls. At elevated concentrations (>1mM) sodium dithionite completely inhibited benzene degradation.However, even at a dithionite concentration of 0.5 mM, the phenoldegradation rates remained unaffected compared to those of unamendedcontrols (data not shown). These results suggest that dithioniteplayed an inhibitory role in the initial hydroxylation stepof benzene degradation rather than the subsequent oxidationof phenol to CO₂ by strain RCB.

H₂O as a potential source of the hydroxyl group.
Previous studies performed using an undefined benzene-degradingmethanogenic enrichment culture also showed that phenol is ametabolite of benzene degradation (36). In these studies, theundefined culture was incubated in media amended with 9% H₂¹⁸O,and a small fraction (2.5%) of the observed phenol pool containedthe ¹⁸O label, suggesting that the hydroxyl group originatedfrom H₂O (36). When a similar experiment was performed by incubatingstrain RCB for 24 h in anaerobic medium prepared using 25% H₂¹⁸O,GC-MS analysis of the ether extracts indicated that there wasan increase of only 1.40% [¹⁸O]phenol in the phenol pool comparedto an unlabeled control culture. A similar increase (1.13% [¹⁸O]phenol)was observed in the phenol pool if the experiment was repeatedusing medium prepared with 50% H₂¹⁸O. The low level of incorporationof the ¹⁸O label into the hydroxyl group of phenol producedfrom benzene under the conditions tested and the lack of a correlationwith the initial H₂¹⁸O content suggest that H₂O is not the sourceof the hydroxyl group during benzene catabolism by strain RCB.This suggestion is supported by the fact that an increase of1.40% [¹⁸O]phenol in the phenol pool was observed in sterileculture medium containing 50% H₂¹⁸O and 1.5 µM unlabeledphenol after 24 h of incubation, demonstrating that the ¹⁸Oatom of H₂¹⁸O could interchange to a small extent with the moreabundant ¹⁶O atom of phenol. This may more likely account forthe [¹⁸O]phenol produced in the biologically active samples.This result is unexpected and leaves the source of the hydroxylgroup unknown. Final elucidation of the details of the mechanisminvolved in the initial hydroxylation process may shed furtherlight on this.

Mechanism of hydroxylation.
In other known microbial hydroxylation reactions involving substratessuch as enoyl-CoA or fumarate, the double bond is generallyadjacent to a carbonyl group that plays a critical role by delocalizingthe negative charge of the reaction intermediate, making thereaction favorable (5). This cannot occur with benzene due toits molecular symmetry and lack of functional side groups. Thus,hydroxyl group addition for benzene under standard conditionsis an endogonic reaction ( ${Delta}$ G^o' = 25.8 kJ/mol) (16). One possiblemechanism for overcoming the unfavorable thermodynamics of benzenehydroxylation is through the formation of highly reactive hydroxylfree radicals (HO·), which subsequently attack the aromaticring (1, 16, 38). When benzene-degrading cultures of strainRCB with nitrate as the electron acceptor were incubated inthe presence of the hydroxyl free radical scavenger sodium iodide(0.5 mM), benzene degradation and subsequent phenol formationwere inhibited compared to controls not amended with sodiumiodide (Table 1). After 24 h of incubation, almost 19 µMof the initial benzene concentration (35 µM) was degradedby active cultures of strain RCB using nitrate as the electronacceptor, while only 1.75 µM of the initial benzene wasdegraded in the samples that were amended with sodium iodide(Table 1). In contrast, sodium iodide addition had no effecteither on phenol degradation by strain RCB under nitrate-reducingconditions or on the growth of strain RCB on acetate and nitrate,demonstrating that the iodide was not generally toxic to thecell (Fig. 6). Phenol degradation by strain RCB proceeded atthe same rate regardless of the presence of 0.5 mM sodium iodide,and almost 50% of the substrate was degraded within 24 h bothin the presence and in the absence of sodium iodide (Fig. 6A).Ion chromatography analysis demonstrated that iodide concentrationsremained constant throughout the incubation, indicating thatthe iodide was not used (oxidized to iodate) in place of benzeneas an alternative, more favorable electron donor by strain RCB(data not shown).

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TABLE 1. Degradation of benzene by strain RCB in the presence and absence of various hydroxyl free radical scavengers

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FIG. 6. Effect of 0.5 mM sodium iodide on phenol degradation by strain RCB (A) and growth of strain RCB with acetate (B). The data are averages of triplicate determinations.

Similar inhibition of anaerobic benzene degradation and phenolformation by strain RCB with nitrate as the terminal electronacceptor was also observed in the presence of other hydroxylfree radical scavengers, such as 10 mM 5,5-dimethyl-1-pyrroline-N-oxideor 10 mM mannitol, which inhibited benzene degradation to differentextents (Table 1). Phenol degradation and acetate oxidationunder nitrate-reducing conditions by strain RCB remained unaffectedin the presence of these scavengers after 24 to 48 h of incubation(data not shown). Although the results described above are notdirect evidence, they strongly support the hypothesis that benzenering hydroxylation is mediated through hydroxyl free radicalattack.

Mechanism of benzoate formation from phenol.
Reports on anaerobic phenol degradation by the monoaromatichydrocarbon degrader Thauera aromatica have demonstrated thatphenol is initially phosphorylated in an ATP-dependent mechanismto form phenylphosphate, which is then carboxylated by phenylphosphatecarboxylase, forming 4-hydroxybenzoate (34). The 4-hydroxybenzoateis further activated by a specific CoA ligase, and the hydroxylgroup is reductively removed (22). Molybdenum-containing iron-sulfurproteins have been shown to catalyze the dehydroxylation ofhydroxy-benzoyl-CoA thioesters (6, 7, 20, 23). Whether a similarmechanism is involved during benzene metabolism by strain RCBis currently unknown. Phenylphosphate or 4-hydroxybenzoate wasnot detected in culture extracts of strain RCB analyzed duringnitrate-dependent benzene degradation. Furthermore, recent reportshave demonstrated that 0.1 mM dithionite inhibits carboxylationof phenylphosphate to form 4-hydroxybenzoate (34). Whether thisis a reversible reaction is not known. In our experiments, whilethe addition of 0.1 mM dithionite to active benzene-degradingcultures decreased the rate of degradation of benzene and thesubsequent formation of phenol (data not shown), it did notcompletely inhibit the process and had no significant effecton phenol catabolism by strain RCB.

Conclusion.
The results presented here provide the first elucidation ofthe initial steps involved in the anaerobic metabolic pathwayfor benzene degradation in pure culture (Fig. 7). These studiesdemonstrated that phenol is a metabolic intermediate of anaerobicbenzene degradation by Dechloromonas strain RCB growing withnitrate as the terminal electron acceptor. Contrary to previousreports on methanogenic benzene degradation in which the sourceof the hydroxyl group of phenol was suggested to be water (36),in strain RCB under nitrate-reducing conditions, phenol formationappears to be mediated through reaction with a hydroxyl freeradical that does not originate from H₂O but is formed on theouter membrane or in the periplasm of the organism. Initialhydroxylation has been suggested to be the first step in degradationof other hydrocarbons, such as naphthalene under sulfate-reducingconditions (3) and recently ethylbenzene under nitrate-reducingconditions (24). The recently purified Mo-containing iron-sulfurprotein that adds a hydroxyl group to ethylbenzene to form (S)-(–)-1-phenylethanolmay provide a model for hydroxylation of benzene (24). The phenolformed from benzene by strain RCB is further carboxylated anddehydroxylated to form benzoate or its CoA ester, benzoyl-CoA.Both phenol and benzoate are formed outside the cytoplasm, andit appears that benzoate is transported into the cell for subsequentcatabolism to CO₂. The environmental implications of such extracellularprocessing of benzene to more readily degradable substratesand the availability of these substrates to other organismsin the natural environment are currently unknown. Although theconcentrations of phenol and benzoate detected in our studiesare very low, they are in agreement with previous reports onintermediates formed during anaerobic benzene and toluene degradation(4, 10). Prior studies detected phenol, cyclohexanone, and propionateas potential intermediates in undefined methanogenic enrichmentsincubated with benzene (21, 37). Similarly, phenol, benzoate,and acetate were also detected in benzene-degrading iron-reducingand sulfate-reducing sediments (10). As Dechloromonas speciesare not known to grow by Fe(III) reduction, by sulfate reduction,or through syntrophic interactions with methanogenic bacteria(13, 17), it is unlikely that organisms closely related to strainRCB or JJ are involved in the benzene metabolism observed inthe previous studies. This, combined with the fact that bothphenol and benzoate have now been identified during anaerobicbenzene degradation in each of these disparate terminal electron-acceptingprocesses, suggests that a single universal biochemical pathwaymay exist for anaerobic benzene degradation and that it is similarto that shown here for Dechloromonas strain RCB. In this regard,strain RCB serves as an ideal model organism to study the biochemistryand genetic regulation of anaerobic benzene oxidation in pureculture.

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FIG. 7. Model of proposed universal pathway of anaerobic benzene degradation. The transient formation of para-hydroxybenzoate (shown in parentheses) is hypothetical.

ACKNOWLEDGMENTS

Studies on Dechloromonas aromatica strain RCB were supportedby independent grants to J.D.C. from the U.S. Department ofEnergy NABIR program (DE-FG02-98-ER-62689) and the U.S. Departmentof Defense (DACA72-00-C-0016).

We acknowledge K. V. Kellaris for iodide analysis in this project.

FOOTNOTES

* Corresponding author. Mailing address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720. Phone: (510) 643-8455. Fax: (510) 642-4995. E-mail: jcoates{at}nature.berkeley.edu.

REFERENCES

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