Cloning and In Situ Expression Studies of the Hydrogenobaculum Arsenite Oxidase Genes

Novel arsenite [As(III)] oxidase structural genes (aoxAB) werecloned from Hydrogenobaculum bacteria isolated from an acidicgeothermal spring. Reverse transcriptase PCR demonstrated expressionthroughout the outflow channel, and the aoxB cDNA clones exhibiteddistribution patterns relative to the physicochemical gradientsin the spring. Microelectrode analyses provided evidence ofquantitative As(III) transformation within the microbial mat.

INTRODUCTION

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INTRODUCTION

We have been studying microbial arsenite [As(III)] oxidationin the outflow channel of an acidic geothermal spring in YellowstoneNational Park with the goal of understanding As biogeochemicalcycling in geothermal environments specifically and, at a moregeneral level, the environmental factors that influence thisimportant microbial process. We have shown that H₂S is a potentinhibitor of microbial As(III) oxidation in this acidic system(3, 5) and that H₂S influences the distribution of an As(III)chemolithotroph within the spring outflow channel (3). However,results from ex situ assays suggested that an As(III) oxidaseenzyme(s) is present in the microbial mat but is presumablymaintained in an inhibited state by H₂S (3). Hydrogenobaculumoverwhelmingly dominates the microbial community inhabitingthis region of the spring outflow channel (>95% of PCR clonelibraries) (4, 8), as well as other acidic geothermal featuresstudied in Yellowstone (14), and consequently, the As(III) oxidaseenzyme(s) of these organisms deserved scrutiny. In this briefcommunication, we describe the cloning, sequencing, and in situexpression studies of Hydrogenobaculum As(III) oxidase genesin this model acidic geothermal spring. We also demonstratethe use of novel microelectrode profiling to identify As(III)and H₂S transformations within the mat.

Cloning of Hydrogenobaculum As(III) oxidase genes.

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Cloning of Hydrogenobaculum...

The research location was Dragon Spring, an acid-sulfate-chloridegeothermal spring, which is a National Science Foundation MicrobialObservatory we have previously described in detail (see references3 and 4 for color images illustrating the outflow channel andrelevant geochemical profiles). Source waters are a nearly constantpH 3.1, temperatures range from 68 to 72°C, and the watercontains roughly 15 nM H₂, $~$ 60 µM H₂S, 33 µM As(III),and 60 µM Fe(II) (4, 9). We focused primarily on the yellowelemental sulfur (S°) deposition zone, though some samplingwas also conducted in the brown Fe-oxyhydroxide zone. Hydrogenobaculumbacteria are present in both (5, 8) but clearly dominate inthe S° zone (4, 8). Ecologically relevant Hydrogenobaculumstrains we isolated from the S° deposition zone (4) wereverified to be capable of oxidizing As(III) (results not shown).The PCR primers used for cloning the aoxAB genes from theseorganisms were aoxFor (5'-TTCGCTGCTTTTTCATTTTG-3') and aoxRev(5'-TGTGATCTCCACAGCATACG-3'), and the PCR conditions used were94°C for 5 min; 30 cycles of 94°C for 1 min, 50°Cfor 1 min, and 72°C for 4 min; 72°C for 10 min; anda hold at 4°C. The 3,496-nucleotide amplicon (GenBank accessionno. FJ376461) included flanking DNA and inferred two peptidesthat were 53 and 35% identical to Herminiimonas arsenicoxydansAoxA and AoxB, respectively. Phylogenetic assessment showedthat Hydrogenobaculum AoxB branched reliably with AoxB fromThermus, another organism deeply rooted in the domain Bacteria(Fig. 1).

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FIG. 1. Phylogeny of Hydrogenobaculum aoxB. Shown is a neighbor-joining-based phylogenetic tree (PAUP 4.0b10) inferring the evolutionary relationship between Hydrogenobaculum aoxB and other aoxB genes within the domains Bacteria and Archaea (accession numbers are in parentheses). Phylogeny is based on alignments of a common 383-amino-acid region (aligned with ClustalW). The tree was rooted with the Escherichia coli formate dehydrogenase (Fdh) (not shown), representing a different clade within the dimethyl sulfoxide reductase family (11). Bootstrap values of >50% are shown.

In situ expression.

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In situ expression.

Acquiring the aox gene sequence provided an essential tool fordetermining whether the Hydrogenobaculum As(III) oxidase genesare being expressed in the high-H₂S regions of the spring system.Total RNA was extracted from the microbial mat at discrete locationsin the S° deposition zone where H₂S levels ranged from 80to 10 µM, concentrations that are consistent with previouschemical analyses (3, 9). The extremely friable nature of themat material made it impossible to collect mat material at specificdepths, and thus a homogenized mat sample was taken at eachtransect site. The RNA extraction and reverse transcriptasePCR (RT-PCR) protocols used were as previously described (1).Primers aoxFor/aoxRev failed to work with all of the mRNA samples,and thus several primer sets representing internal sectionsof Hydrogenobaculum aoxB were examined. RT-PCR primers aoxBRTFor(5'-CAAGGAGTAGCCCTGCAAAG-3') and aoxBRTRev (5'-CCTTGAAGGTTTGGCATCAT-3')(94°C for 5 min; 30 cycles of 94°C for 45 s, 50°Cfor 45 s, and 72°C for 1 min; 72°C for 10 min; and ahold at 4°C) were found to consistently yield the expectedamplicons (593 nucleotides), regardless of H₂S(aq) (Fig. 2).PCR controls were negative, demonstrating that there was noDNA contamination (Fig. 2).

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FIG. 2. In situ expression of Hydrogenobaculum aoxB. Shown is an agarose gel illustrating the RT-PCR amplicons obtained from RNA extracted from regions of the Dragon Spring outflow channel that vary in H₂S concentration (the Fe-oxyhydroxide brown mat zone is represented by 0 H₂S). For each lane, 10% of the PCR amplicon was loaded. MW, molecular size.

Sequencing of cloned RT-PCR amplicons revealed a range of uniqueaoxB sequences (GenBank accession no. FJ376462 to FJ376483)with 92 to 99% amino acid identity with the aoxB genes clonedfrom the pure Hydrogenobaculum cultures and represented 22 differentinferred amino acid sequence variants from the S° and Fe-oxyhydroxidezone sediments. Eleven cloned cDNAs (clone Dr5.1) were identicaland only found near the spring source (5 cm). Given their clearphylogenetic relatedness to Hydrogenobaculum relative to theaoxB genes of other defined taxa (Fig. 3), the Dr5.1 sequencesmay be evidence of distinct Hydrogenobaculum population distributionpatterns. cDNA clone sequences derived from the S° depositionzone clustered with that of the pure Hydrogenobaculum culturesbut were separate from those of the brown Fe-oxyhydroxide zone(Fig. 3). The aoxBRTFor/aoxBRTRev primers failed to amplifythe aoxB gene from the As(III) chemolithoautotroph Acidicalduswe previously isolated from the brown mat (3), implying thatthe brown mat amplicons were from an organism(s) that is distinctfrom Acidicaldus. Phylogenetic comparison with aoxB sequencesavailable from other pure-culture-characterized organisms suggeststhat if these particular cDNA clones are not from a Hydrogenobaculumsp., then they could likely derive from a closely related taxonwithin the phylum Aquificae. Unifrac analysis (10) supportedthe view that the S° deposition zone and the brown Fe-oxyhydroxidezones were different environments with respect to aox gene diversity(raw P score, <0.0001).

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FIG. 3. AoxB diversity in Dragon Spring. Amino acid sequences were inferred from aoxB cDNAs amplified from the Dragon Spring yellow S° deposition zone and brown Fe-oxyhydroxide mat zone. Relative phylogeny was estimated by using the neighbor-joining algorithm in PAUP 4.0b10, with comparisons based on a 183-amino-acid region corresponding to aoxB nucleotide positions 262 to 684. Dragon clone numbers correspond to distances from the source and clone designators. For clones represented multiple times in the library, the number in parentheses after the clone number indicates the number of clones containing this sequence. Other aspects of this comparison not shown (to conserve space) include all of the other sequences illustrated in Fig. 1 and for which all elements of topology were completely preserved.

Microelectrode studies.

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Microelectrode studies.

We employed microelectrode technology for real-time simultaneousmeasurement of H₂S and As(III) across small vertical spatialscales in the microbial mat, with construction and use of thegold amalgam microelectrode as previously described (2, 7).H₂S and As(III) concentrations in the overlying geothermal waters(Fig. 4) were completely consistent with previous measurementsby traditional aqueous chemistry (3, 4, 9). However, as theamalgam microelectrode was incrementally lowered into the S°mat, As(III) levels declined sharply at approximately the 1-mmdepth and corresponded to a similarly sharp decline in H₂S (Fig.4).

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FIG. 4. Vertical As(III) and H₂S gradients in the Dragon Spring microbial mat. As(III) ( ${circ}$ ) and H₂S (•) concentrations were estimated by cyclic voltammetry. For orientation, measurements were initiated at the water surface and the dashed horizontal line represents the approximate upper boundary of the microbial mat. The H₂S data were previously reported by D'Imperio et al. (4).

Conclusions.

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Conclusions.

The results of experiments summarized herein provide severalnew and key elements to our understanding of microbial As(III)oxidation in geothermal environments. The ability to clone theHydrogenobaculum aoxAB genes provided an opportunity to linkan aox sequence with a specific cultivated and characterizedmicroorganism outside the phylum Proteobacteria and representsan incremental but important expansion of a very limited aoxphylogeny database. The relatedness of Hydrogenobaculum aoxBto that from Thermus is consistent with previous observationsthat the phylogenetic relationships of genes encoding arsenicmetabolism are similar to 16S rRNA gene phylogeny and thus suggestsa long evolutionary history (6). Further, the distinct distributionpattern of the novel cDNA clones correlated with equally distinctgeochemical gradient zones where we have documented H₂S andH₂ to be present (S° deposition zone) or below detection(brown Fe-oxyhydroxide zone) (3, 4). Documentation of aoxB expressionin the S° deposition zone provides direct evidence thatAs(III) oxidase genes are indeed being expressed and affirmsour prior work, in which ex situ assays suggested that As(III)oxidases are present in the mat but inactive due to H₂S inhibition(3). Studies concerning H₂S inhibition of As(III) oxidationin pure cultures of the aerobic As(III) chemolithoautotrophAcidicaldus isolated from this spring established that the H₂Sinhibition was uncompetitive in nature and not due to H₂S consumingO₂, the electron acceptor for As(III) oxidation.

Microelectrode data obtained in the present study representnovel observations with respect to environmental arsenic biogeochemistry.These real-time in situ geochemical determinations illustrateda sharp and nearly quantitative decline in As(III) and H₂S concentrationsas a function of mat depth (Fig. 4), clearly suggesting thatboth are subjected to important transformations within the matover very small spatial scales (Fig. 4). The similarity of theseprofiles is consistent with previous work showing the dependenceof As(III) oxidation on the absence of H₂S (3, 5), though inthis study the 50-µm steps used for microelectrode measurementswere too coarse to allow us to identify the exact H₂S concentrationat which the As(III) transformation occurred in the verticaldimension. We note, however, that the highly friable natureof the S° flocs renders the collection of biomass (for RNAextraction) from specific depths with 50-µm spatial resolutionvery difficult, and thus we were not able to examine aox expressionat this fine spatial scale to determine if aoxB expression verticallywithin the mat may also be correlated with H₂S levels.

The disappearance of the H₂S signal is consistent with its oxidationby the microaerobic Hydrogenobaculum bacteria known to dominatethe S° deposition zone and that we have shown to grow withH₂S as an energy source (4). The basis for the nearly quantitativeloss of the As(III) signal may be biotic or abiotic. Transformationproducts would likely not be thioarsenites, as the latter arealso electroactive with Au amalgam electrodes (G. K. Druschel,unpublished data), react at the same potential as As(III) inorganicspecies, and were concluded to be absent by Planer-Friedrichet al. (13). Rather, the loss of the As(III) signal likely indicatesthe formation of the arsenate oxyanion or thioarsenates, neitherof which are electroactive at Au amalgam electrodes and thusaccount for the loss of signal. Arsenate is the major transformationproduct observed in previous ex situ assays conducted with thismat material (3, 9), and thioarsenates have been reported tooccur in the S° deposition zone of this spring (13). Methylatedarsenicals are also possible and have also been detected inthis spring, though it is unclear at what concentration (12).One feature common to these latter arsenic compounds is thatthey all represent oxidation products; initial attempts to detectreduced volatile species at this site (e.g., AsH₃) have thusfar been unsuccessful (12). As abiotic formation of thioarsenatesis favored under high-pH conditions (15), it will be interestingto determine whether their occurrence in this acidic environmentderives directly from microbial activity.

ACKNOWLEDGMENTS

This study was supported primarily by the National Science FoundationMicrobial Observatories Program (MCB-0132022 to T.R.M.). Additionalsupport was from the National Aeronautics and Space Administration(NAG 5-8807), the Montana Agricultural Experiment Station (911310),and the Gordon and Betty Moore Foundation (to T.R.M.). Supportfrom the American Chemical Society Petroleum Research Fund (43356-GB2to G.K.D.) is also acknowledged. The Thermal Biology Institute,Montana State University, Bozeman, funded a summer visitingscholarship for G.K.D. during part of this study.

We all thank John Varley and Christie Hendrix at the YellowstoneCenter for Resources, Yellowstone National Park, WY, for assistancein permitting.

FOOTNOTES

* Corresponding author. Mailing address: Department of Land Resources & Environmental Sciences, Montana State University, Bozeman, MT 59717. Phone: (406) 994-2190. Fax: (406) 994-9333. E-mail: timmcder{at}montana.edu

Published ahead of print on 20 March 2009.

REFERENCES

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