This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kashyap, D. R.
Right arrow Articles by McDermott, T. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kashyap, D. R.
Right arrow Articles by McDermott, T. R.

 Previous Article  |  Next Article 

Journal of Bacteriology, February 2006, p. 1577-1584, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1577-1584.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Na+:H+ Antiporter and a Molybdate Transporter Are Essential for Arsenite Oxidation in Agrobacterium tumefaciens

Des R. Kashyap,1 Lina M. Botero,1 Corinne Lehr,1 Daniel J. Hassett,2 and Timothy R. McDermott1*

Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana 59717,1 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-05242

Received 12 August 2005/ Accepted 4 December 2005


arrow
ABSTRACT
 
Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na+:H+ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genome-sequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na+ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.


arrow
INTRODUCTION
 
Microbe-arsenic interactions are viewed as a major driver of arsenic (As) chemical speciation in nature, which in turn is a critical factor in determining As fate and transport in the environment (reviewed in references 19 and 38). Many types of As transformations have been documented in various microorganisms (6, 14, 18, 34, 41, 47), although those currently seen to dominate As speciation in the environment involve either arsenite [H3AsO3, As(III)] oxidation or arsenate [HAsO42–, As(V)] reduction. These redox transformation reactions are generally thought to be used by microorganisms either for detoxification or for generating cellular energy to support growth (see reviews in references 19, 38, 49 and 50).

Detoxification-based As(V) reduction has been documented to occur in microorganisms throughout the domains Bacteria and Archaea (38, 49), and involves As(V) reduction to As(III) via an As(V) reductase, with the As(III) then extruded by the ArsB efflux pump that is efficient at removing As(III) and antimonite [Sb(III)]. This process, as well as the genes encoding the enzymes and regulatory proteins involved, has been extensively studied and recently reviewed by Silver and Phung (49). Dissimilatory As(V) reduction has also been documented to occur in numerous prokaryotes in both prokaryotic domains (38), although its original discovery was more recent (2), and as a consequence, far less is known about the genetic determinants required for anaerobic As(V) respiration. Dissimilatory As(V) reductases from Chrysiogenes arsenatis (25) and Bacillus selenitireducens (1) have been purified and characterized, and the arr genes have been identified in Shewanella sp. strain ANA-3 (45).

Microbial As(III) oxidation was first reported by Green (16) and then later by several labs studying a variety of organisms (e.g., references 10, 14, 15, 39, 41, 44, and 57). Initial reports of microbial As(III) oxidation concerned heterotrophs, and thus, more or less by default, As(III) oxidation in these organisms was viewed as a detoxification strategy. This is due to the fact that As(V) is less toxic than As(III) (7, 42), which has a strong affinity for protein sulfhydryl groups (22, 24). The As(III) oxidase enzyme was first purified from the heterotroph Alcaligenes faecalis and characterized biochemically (4), its amino acid sequence was determined, and its crystal structure was characterized (12). The structural genes coding for this heterodimeric enzyme were recently cloned from Cenibacterium arsenoxidans (35) and named aoxA (small subunit, Rieske type) and aoxB (large subunit). C. arsenoxidans aox mutants were found to be more sensitive to As(III) than the wild-type parental strain (35), providing the first evidence that As(III) oxidation may indeed play a detoxification role.

As(III) chemolithotrophy has been reported in an Agrobacterium/Rhizobium-like {alpha}-proteobacterium referred to as NT-26 (47) and in a facultative anaerobic {gamma}-proteobacterium isolated from the hypersaline Mono Lake (37). The genes encoding the As(III) oxidase in NT-26 were cloned and assigned different mnemonics, aroA and aroB for the large and small subunits, respectively (48). This As(III) oxidase has also been purified and partially characterized (48).

Other than the structural genes for As(III) oxidase, there is currently no information available that describes other loci that encode other functions important to As(III) oxidation. Several heterotrophic As(III)-oxidizing soil microorganisms were recently isolated (29). One of these isolates was identified as Agrobacterium tumefaciens as determined by sequencing the near-full-length 16S rRNA gene (1,400 nucleotides [nt]) (29). We used this genetically tractable soil isolate, strain 5A, as a model for further investigations of the genetics and physiology of As(III) oxidation in bacteria. In this report, we describe results from a mutagenesis study that identified two new loci encoding functions that are essential for As(III) oxidation in this organism.


arrow
MATERIALS AND METHODS
 
Bacterial strains and media. Bacterial strains and plasmids used in this study are listed in Table 1. A. tumefaciens strains were grown in either a defined minimal mannitol medium (MMN, amended to 30 µM phosphate) (52) or Luria-Bertani medium (LB) (46). Escherichia coli strains were always cultured in LB. Gentamicin (25 µg · ml–1) was added for selection of transposon transconjugants, and tetracycline (10 µg · ml–1) and ampicillin (100 µg · ml–1) were included for selection of transformants when (sub)cloning PCR amplicons or when mobilizing plasmids into A. tumefaciens mutants (described below). As(III) (as NaH2AsO3) or As(V) (as NaH2AsO4) was included at designated levels to examine As transformation phenotypes, and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal; 40 mg · liter–1 agar medium) was added as required for detection of PCR amplicons cloned into either pTA2.1 or pCR-XL-TOPO or subcloned into pRK311.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Bacterial strains and plasmids used in this study

Transposon mutagenesis and detection of As(III) oxidation mutants. Transposon mutagenesis was accomplished using transposon Tn5-B22 (51) with E. coli strain S17-1 as the conjugal donor and was conducted as previously described (52). Putative As(III) oxidase mutants were identified by subculturing (in duplicate) transconjugants onto MMN-gentamicin agar amended with 1 mM As(III). One set of plates was flooded with 0.1 M AgNO3 (27) to detect the presence of As(V) associated with As(III)-oxidizing transconjugants (dark brown colonies) or the absence of As(III) oxidation (yellow colonies), which would imply a mutant incapable of oxidizing As(III). Yellow colonies were subcultured to purity, and the mutant phenotype was verified with analytical techniques that directly measured As(V) formation (see below).

The transposon insertion sites of unique mutants were characterized using an arbitrary PCR approach employing the APAgene genome walking kit (Bio S&T Inc., Montreal, Quebec, Canada). Forward primers for the first, second, and third consecutive PCRs in this protocol were 5'-GGCGACGTTAACCAAGCGGGCAGTACGGC-3', 5'-GCCCAGTCGGCCGCACGATGAAGAGCAG-3', and 5'-GGAAAACGGGAAAGGTTCCGTTCAGGACGC-3', respectively. These primers represent sequences that are unique to the transposon, are progressively closer to the extreme end of the transposase arm, are separated by 67 to 80 nucleotides, and were designed to amplify the genomic DNA immediately flanking the transposon. The reverse primers were supplied by the kit manufacturer, and the PCR conditions were as described in the protocols supplied by the manufacturer. Mutant chromosomal DNA served as template for the first round of PCR, and then amplicons from the first and second PCRs served as template for the second and third PCRs, respectively. The products from the third PCRs were separated by electrophoresis in 1.0% low-melting-point agarose gels in Tris-acetate-EDTA buffer, with the brightest and highest-molecular-weight band excised, followed by DNA extraction and purification using Agar ACE agar-digesting enzyme (Promega, Madison, WI). The purified PCR product was adenylated with Taq polymerase (Promega) and then cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and transformed into competent E. coli strain TOP10 (Invitrogen). Gene identification was based on sequencing of the cloned amplicons, using an ABI310 DNA sequencer (Applied Biosystems, Norwalk, CT) and synthetic primers complementary to the vector plasmid sequences flanking the multiple cloning site (TOPOF, 5'-TCTAGATGCATGCTCGAGCGG-3', and TOPOR, 5'-CCAAGCTTGGTACCGAGCTCG-3') and to internal sequences. Homology searches of public databases were conducted using BLAST (3), and sequence alignments were completed using ClustalW (56).

Mutant complementation. Operons containing homologues of the genes identified in the mutants were found in the genome sequence of A. tumefaciens strain C58 (GenBank AE008688) and PCR cloned from C58 genomic DNA for complementation experiments. Standard PCR cloning procedures were then employed to clone the targeted operons along with flanking DNA into pCR-XL-TOPO (Invitrogen). Primer design for this cloning work included BamHI restriction sites that allowed each cloned amplicon to then be subcloned as BamHI fragments into the broad-host-range cosmid pRK311 (Table 1). Sample sequencing reactions verified the identity of the cloned fragment, and all (sub)cloning work used standard molecular biology protocols described by Sambrook et al. (46). Transformations used chemically competent cells and standard techniques also as described by Sambrook et al. (46). Recombinant plasmids were mobilized into each mutant using E. coli S17-1 as the single donor strain, employing techniques that we have described previously (32).

Gene induction experiments. Reverse transcriptase PCR (RT-PCR) and ß-galactosidase reporter gene assays were used to study As(III)-dependent expression of the mod and mrp operons, respectively. In both cases, the wild-type strain 5A was cultured in the presence or absence of 100 µM As(III). ß-Galactosidase assays were performed as we have previously reported (53) and were an option for the mrp mutant because Tn5-B22 was in the correct transcriptional orientation relative to mrpB so as to allow the promoterless lacZ gene carried by the transposon to report mrpB expression. For RT-PCR experiments, samples of 5A cultures taken for total RNA extraction were transferred to cold centrifuge tubes and diluted with 10 ml ice-cold 0.85% NaCl containing 40 µg · ml–1 chloramphenicol. The cell suspension was centrifuged at 12,000 x g for 7 min at 1°C, and the supernatant was discarded. The cell pellet was resuspended in 400 µl of the saline-chloramphenicol solution, transferred to a chilled microcentrifuge tube, and centrifuged again at 13,000 x g for 4 min at 4°C. The supernatant was discarded, and the cell pellet was resuspended in nuclease-free water (Promega), snap frozen in liquid nitrogen, and stored at –75°C.

Total RNA was extracted from cells using a protocol that we have previously applied to environmental samples (36). RNA was treated with DNase (Promega) and then purified using the Ambion (Austin, TX) MEGAclear kit following the manufacturer's instructions. DNA was verified to be absent by PCRs with PCR mixtures containing 50 ng of RNA preparation, Tfl DNA polymerase (Promega), and 0.4 µM of each primer of the modB gene (forward primer, 5'-CTTGTGTATAAGAGTCAGCCC-3'; reverse primer, 5'-GACGATTGTGGGATTATGGCT-3'). RT-PCRs were conducted using the Access Quick RT-PCR system (Promega), including 0.4 µM each of the modB primers and 50 ng of total RNA. The RT-PCR protocol consisted of 45 min at 48°C; 94°C for 2 min; and 30 cycles of 94°C for 30 s, 55°C for 1 min, and 68°C for 2 min. The final extension was 7 min at 68°C, and RT-PCR products were sample sequenced to verify the identity of the amplicon.

As(III) sensitivity. To assess the As(III) sensitivity of the mutants, cultures were grown to late log phase in MMN broth and then inoculated (starting A595= 0.1) into fresh MMN containing 0 mM, 0.5 mM, 1.0 mM, 2 mM, or 3 mM As(III). After 24 h of incubation (30°C with shaking on an orbital shaker water bath), culture optical density was measured (A595).

Arsenic analytical chemistry. Analytical As chemistries were determined using techniques previously described (10). Briefly, cell suspensions were centrifuged and supernatants were filtered (0.22-µm pore size) into two separate 15-ml bottles (5 ml each). The first was acidified with 0.1 ml of 12.1 M HCl and stored at 4°C until analyzed for total As [As(ts)]. As(V) was determined in the second aliquot by measuring total As after removing As(III) by treatment with 1.0 ml of 2 M Tris buffer (pH 6) and sparging with N2 while 1 ml of 3% (wt/vol) NaBH4 (in 0.1% NaOH) was added in 0.2-ml increments over 4 min. Samples were then sparged for an additional 3 min, acidified with 0.1 ml of 12.1 M HCl, and stored at 4°C. As(III) concentration was determined by difference between total As(ts) and As(V).

Nucleotide sequence accession numbers. GenBank accession numbers are DQ298020 for the mrpB homologue, DQ309024 for the region corresponding to the 5' end of mrpD, and DQ298021 for the region spanning the junction between mrpD and mrpE, and DQ351525 for modB.


arrow
RESULTS
 
Identification of As(III) oxidation mutants. Screening of the transposon mutants with silver nitrate staining on MMN agar amended with 30 µM phosphate and 1 mM As(III) identified several A. tumefaciens 5A Genr transconjugants that were negative for As(III) oxidation (results not shown). Southern blot probing of total genomic DNA digests (BamHI and EcoRI digests) with a 32P-labeled internal transposon fragment identified likely siblings (results not shown) and allowed us to focus subsequent characterization work on unique mutants.

Arbitrary PCR anchored with the transposase-specific primer verified that the Tn5-B22 insertion site was different for each of two mutants that were the subject of this study (Fig. 1). The transposon insertion in mutant MSUAt2 (Fig. 1A) interrupted a homologue to an open reading frame (ORF) in Agrobacterium tumefaciens strain C58 annotated in the genome sequence (NP_531608) as mnhB (93% identity/97% similarity; 137 amino acids). This type of antiporter is also referred to as pha in Sinorhizobium meliloti, sha in Bacillus subtilis, and mrp in Bacillus halodurans (recently reviewed in reference 55). We have elected to use the mrp mnemonic because of its overall more extensive usage (55). Additional primer walking experiments and partial sequencing in this region of the 5A genome revealed the presence of adjacent ORFs showing similarly high homology to mrpD and mrpE (Fig. 1A), implying a similar mrp gene arrangement in the A. tumefaciens strain being studied.


Figure 1
View larger version (10K):
[in this window]
[in a new window]
  		FIG. 1. Cartoon illustration characterizing the Tn5-B22 insertions in each mutant and the genomic DNA PCR cloned from A. tumefaciens strain C58 for complementation experiments. For each mutant, the gray arrows represent specific genes and their orientation as they occur in the A. tumefaciens C58 genome and as they were PCR cloned into their respective mutants. Small horizontal bars underneath the gray arrows indicate regions of the respective mutant genome that were sequenced via primer walking experiments. (A) Tn5-B22 insertion in an mrpB homologue (annotated as mnh in strain C58). Point of insertion was estimated to be at nt 8 based on nucleotide alignments with the C58 strain mrpB/mnhB. (B) Tn5-B22 insertion that approximately bisects the modB gene (based on alignment with the A. tumefaciens C58 modB). Point of insertion was estimated to be at nt 299 based on nucleotide alignments with the C58 strain homologue. Tn5-B22 insertion points are indicated by the inverted arrowheads. Positions of the priming sites (identified in Results) used for amplification are shown.

The Tn5-B22 insertion in a second mutant, strain MSUAt6 (Fig. 1B), was found to approximately bisect an ORF (based on sequence alignments) coding for an inferred amino acid sequence having high identity (74 to 98% across 100 amino acids) to predicted ModB proteins in several organisms, in which it serves as the transmembrane permease component of a high-affinity, ABC-type, molybdate transport system (5, 28).

Mutant complementation. The importance of the mod and mrp operons for As(III) oxidation was verified with complementation experiments. The A. tumefaciens C58 genome contains apparent operons annotated as mnh (mrp used here as explained above) and mod operons that share significant homology with the genes in question. They were PCR cloned and mobilized into MSUAt2 and MSUAt6, respectively. For MSUAt2, the entire C58 mrp/mnhABCDEFG operon (6,734 bp), along with 507 bp upstream and 279 bp downstream (primers P2F and P2R, Fig. 1A), was PCR cloned into pCR-XL-TOPO and then subcloned into pRK311 as a 7,472-bp BamHI fragment to generate pLB403. When conjugated into MSUAt2, pLB403 restored As(III) oxidation (Fig. 2A). The same PCR strategy (using primers P6F and P6R, Fig. 1B) was employed to clone the entire C58 modABCE operon into pRK311 (to form pLB404), which was then mobilized into MSUAt6 (as a 4,049-bp BamHI fragment). The C58 mod operon complemented the mutation in MSUAt6 (Fig. 2B). Both mutants containing the control plasmid pRK311 remained negative for As(III) oxidation (Fig. 2A and 2B).


Figure 2
View larger version (25K):
[in this window]
[in a new window]
  		FIG. 2. Complementation of the As(III)-oxidase mutants. (A)MSUAt2. (B) MSUAt6. Culture growth is shown with closed symbols, and As(V) concentration is shown with open symbols. Symbols: squares and circles, MSUAt2 or MSUAt6; triangles, MSUAt2(pLB403) or MSUAt6(pLB404); diamonds, MSUAt2(pRK311) or MSUAt6(pRK311). Results are from one of two independent experiments demonstrating complementation. Error bars, where visible, represent 1 standard error of the mean calculated from two replicate cultures. Starting As(III) concentration was 50 µM.

Mutant characterization. (i) As(III) tolerance. The isolation of these mutants offered the opportunity to determine whether As(III) oxidation in 5A served a detoxifying function. At As(III) concentrations ranging from 0 to 3 mM, neither of the mutants appeared significantly different from the wild-type strain with respect to As(III) tolerance. As determined by measuring culture optical density after 24 h of incubation, all strains appeared to grow nearly normally in the presence of 0.5 mM As(III) but then displayed progressively poorer growth at 1, 2, and 3 mM As(III) [20%, 80%, and 95% growth inhibition relative to zero As(III), respectively].

(ii) NaCl and LiCl sensitivity of the mrpB mutant. Experiments were then conducted to determine whether the mrp operon in 5A is important for Na+ efflux as was demonstrated in mrp/mnh mutants of Staphylococcus aureus (17) and B. subtilis (20). Incubation of 5A, MSUAt2, and MSUAt2(pLB403) in LB broth modified to contain various amounts of NaCl or LiCl (0 to 0.5%, wt/vol) showed the mutant to be exquisitely sensitive to both salts, whereas pLB403 restored the normal growth response to that of the wild-type strain (Fig. 3).


Figure 3
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3. Salt tolerance of A. tumefaciens as affected by the mrpB::Tn5-B22 mutation. (A) NaCl. (B) LiCl. Wild-type strain, {square}; mutant MSUAt2, {blacksquare}; mutant MSUAt2 complemented with the cloned C58 mrp/mnhABCDEFG operon (pLB403), •. Cultures were grown in modified LB broth amended with various amounts of NaCl or LiCl as shown. Results are the mean ± 1 standard error averaged from single cultures from two independent experiments (error bars are hidden by the symbols).

(iii) As redox transformation phenotype. Our previous work showed that, even though the wild-type strain exhibited an As(III)-oxidizing phenotype, it nevertheless contained an arsC gene (29), which codes for As(V) reductase. Therefore, we investigated both mutants to determine if the loss of As(III) oxidation capability would reveal an As(V) reduction phenotype. This was indeed the case (Fig. 4). AgNO3 staining of MMN agar containing 1 mM As(V) and inoculated with either mutant or with either mutant carrying the control plasmid pRK311 demonstrated As(V) reduction. In contrast, no As(V) reduction was apparent with the wild-type strain 5A or either mutant carrying its respective complementing PCR-cloned genes (Fig. 4).


Figure 4
View larger version (56K):
[in this window]
[in a new window]
  		FIG. 4. AsV reduction phenotype of the mrpB mutant. Shown is the AgNO3 staining phenotype of MSUAt2 on MMN agar containing 1 mM As(V). The presence of As(V) is indicated by dark brown, whereas the presence of As(III) is indicated by yellow. Results with MSUAt6 were identical and are not shown.

(iv) modB and mrpB transcription. RT-PCR experiments were conducted using RNA harvested from As(III)-exposed and As(III)-naïve cells to determine whether the mod operon is regulated by As(III). The RT-PCRs generated a single modB cDNA product (amplicon sequence verified) from RNA extracted from As(III)-treated cells but not from RNA taken from As(III)-naïve cells (Fig. 5A). As controls, RT-PCRs using primers specific for 16S rRNA yielded strong products of the expected size (1,395 bp) for both culture treatments. In contrast, the mrpB::lacZ reporter assays suggested no As(III) regulatory effects on the mrp operon (Fig. 5B). There were no significant differences in reporter enzyme activity levels between MSUAt2 cultures exposed to As(III) and cultures incubated without As(III). Reporter enzyme levels in the mutant were, however, very significantly greater than background levels in the wild-type strain (Fig. 5B).


Figure 5
View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5. Expression of modB and mrpB in response to As(III) exposure. (A) RT-PCR cDNA amplified from wild-type strain 5A RNA. Lane assignments: lane 1, molecular mass standards; sizes in kilobases are noted to the left of image; lane 2, early-log-phase, As(III)-naïve cells; lane 3, As(III)-exposed cells; lanes 4 and 5, amplicons derived from RT-PCRs using primers specific for the 16S rRNA as internal controls and corresponding to lanes 2 and 3, respectively. (B) mrpB::lacZ reporter gene activity recorded with MSUAt2 (closed symbols) and wild-type 5A (open symbols). Reporter enzyme units are {Delta}A415 · min–1 · culture optical density (measured as A595)–1. Cultures were incubated without As(III) (squares) or with 100 µM As(III) throughout the entire incubation (triangles) or spiked with 100 µM As(III) at mid-log phase (circles). Error bars represent 1 standard error of the mean calculated from triplicate cultures. Some standard errors are hidden by the symbols.


arrow
DISCUSSION
 
Recent studies have documented the cloning and characterization of the structural genes coding for As(III) oxidases in a heterotroph (35) and an As(III) chemolithoautotroph (48). In the current study, we identified additional genes that are required for As(III) oxidation in A. tumefaciens strain 5A. Strain 5A was originally isolated from an As(III)-treated soil column along with another A. tumefaciens isolate that was phylogenetically identical (across 1,400 nt of the 16S rRNA gene) but which displayed an As(V)-reducing phenotype (29). Initial investigations using repetitive extragenic palindromic PCR and internal transcribed spacer PCR techniques suggest that these two A. tumefaciens isolates are highly related (results not shown), but at present we are uncertain as to the exact nature of their relatedness. For example, it is not yet clear if the As(V)-reducing strain acquired the capacity to oxidize As(III) and thus yielded the 5A phenotype, or conversely if the As(V)-reducing isolate actually represents another 5A mutant that has lost the capacity to oxidize As(III) (e.g., point mutation). Initial gene probing and PCR experiments revealed that strain 5A contained an arsC gene and thus this organism has the basic genetic determinants required for As(V) reduction (29). In the present study, we showed that, when mutations eliminate As(III) oxidation, the organism is converted to an As(V)-reducing phenotype (Fig. 4). That this resulted from presumably nonregulatory mutations in two different loci coding for distinctly different functions suggests that this common result is not an artifact. Our other recent work with this organism has identified an operon that contains the As(III) oxidase structural genes as well as regulatory genes coding for a putative two-component signal transduction system that appears to be required for As(III) oxidation (23). We have found this other locus to be up-regulated by As(III) and thus in this respect consistent with the well-established regulatory controls of ars-encoded As detoxification. Assuming that arsC in 5A is controlled in a similar fashion, then this implies that genes encoding functions necessary for both redox transformations may be occurring simultaneously in this organism but that the rate of As(III) oxidation exceeds that of As(V) reduction, with the net outcome being an As(III)-oxidizing phenotype. It is unknown how prevalent such a dual genotype may be among bacteria in the environment; however, the discovery of an organism having the potential to simultaneously reduce As(V) and oxidize As(III) is the first such discovery and serves notice that microbe-As interactions in nature may turn out to be quite complex. An examination of the numerous genome sequences currently available (GenBank) suggests that the capacity to reduce As(V) as encoded by arsC is widespread, and thus, it would seem possible that organisms exhibiting an As(III)-oxidizing phenotype may be similar to strain 5A in this regard.

The function of As(III) oxidation in strain 5A is, at present, enigmatic. This particular organism was isolated as a heterotroph growing on glucose but apparently cannot couple As(III) oxidation to the generation of cellular energy for growth (29). Experiments in the current study pursued this issue further, assessing whether As(III) oxidation may serve as a detoxification mechanism as was reported for Cenibacterium arsenoxidans (35). We were unable to demonstrate increased As(III) sensitivity in the mutants isolated in this study, although it is important to note that the screening methodology selected for mutants that were tolerant of 1 mM As(III) [lower As(III) concentrations in the AgNO3 screening technique gave inconsistent staining results on MMN agar medium (results not shown)]. This may have biased our results by eliminating mutants that were exquisitely As(III) sensitive (e.g., as we have found with Pseudomonas aeruginosa [40]), but the fact remains that the complete loss of As(III) activity due to non-regulatory-type mutations did not yield an As(III)-sensitive phenotype.

Mutant characterization. The Mo requirement of As(III) oxidase (12) is consistent with the As(III) oxidase phenotype of mutant MSUAt6, where the transposon was found to have interrupted a high-affinity molybdate transport operon. We have identified the As(III) oxidase structural genes in strain 5A (23) and found them to be nearly identical to those described for the As(III) chemolithoautotroph (47), including that region intimately associated with where crystal structure studies (12) predict Mo to be located in the native enzyme. Mobilizing the modABCE genes from the genome-sequenced A. tumefaciens strain C58 into MSUAt6 reverted the mutant to the As(III)-oxidizing phenotype of the parent strain (Fig. 2B) and confirmed the importance of these genes and the requirement of Mo. The essential requirement of Mo for the As(III) oxidase is also consistent with As(III)-sensitive transcriptional control of these genes (Fig. 5A) and is of particular significance because this would then ensure adequate Mo supply for the As(III) oxidase enzyme and reduce reliance on regulatory control based on Mo availability (11), which may not necessarily coincide with As(III) exposure levels. To further explore this issue, we amended the MMN agar with 50 µM Mo (as Na2MoO4 · 2H2O) and found that the As(III) oxidase defect could be reversed in the absence of the C58 modABCE genes (results not shown). This then suggests that an alternative Mo uptake system is functional in this organism and is capable of facilitating Mo acquisition when Mo concentrations are relatively high (i.e., a low-affinity uptake system).

Clearly most intriguing was the discovery that a Na+:H+ antiporter is somehow involved in As(III) oxidation. MSUAt2 was very sensitive to NaCl and LiCl and thus is consistent with the phenotype of the S. aureus mnh and B. subtilis mrp mutants (17, 20), and this clearly implies that in A. tumefaciens the Mrp complex is involved in Na+ circulation across the cytoplasmic membrane. However, the contribution of this antiporter to As(III) oxidation is not clear at this juncture. Potentially, it could be involved in As(III) movement across the cytoplasmic membrane. Meng et al. (33) recently provided evidence that ArsB can behave as a metalloid:H+ antiporter, facilitating Sb(III) transport across the membrane for removal from intact cells. In preliminary experiments, we examined As(III) accumulation in wild-type and MSUAt2 cells to assess the unlikely possibility of Mrp facilitating As(III) uptake. Using nonradioactive As(III), nitric acid extraction, and measurement of total cellular As contents, we found no difference between mutant and wild-type cells. However, given the presence and activity of an As(V) reductase and resistance to high levels of As(III), we assume that this organism has an arsB gene, encoding the As(III) efflux pump, which could mask As(III) uptake capacity by whatever mechanism.

Mrp-type Na+:H+ antiporters are structurally complex with distinctive properties relative to other cation/proton antiporters (21), resulting in their separate classification (43). Based on annotated genomic sequence, Mrp antiporters are widespread throughout the phylogenetic tree (55). A collection of experimental evidence in the literature suggests that Mrp-type antiporters have features that may be relevant to the role of the Mrp antiporter in As(III) oxidation. Subunits MrpA, MrpC, and MrpD share significant homology (~20% identity/~40% similarity) with elements of proton-translocating NADH:quinone oxidoreductases in bacteria and mitochondria (13, 17, 26, 30, 31), leading to the suggestion that this complex type of the antiporter may use primary redox energy to directly energize antiport activity (55). Such suggestions stem from observations documenting significantly enhanced nonfermentative growth of an E. coli NADH dehydrogenase ({Delta}nuo {Delta}ndh) mutant when transformed with a cloned Bacillus Mrp antiporter (54). No such growth enhancement was observed when the same mutant was transformed with NhaA, a structurally much simpler and separate type of secondary Na+/H+ antiporter (20). Furthermore, the growth stimulation of this mutant persisted in a mutated Mrp that is deficient in Na+ efflux (54) with one implication then being that Mrp antiporters may have additional functions that are separate from controlling Na+ flux across the cytoplasmic membrane. Additional experimental evidence suggested that the capacity of the cloned Mrp operon to rescue the growth defect was linked to increased activity of a malate:quinone oxidoreductase of the mutant E. coli host (54). An electron transport-based mechanism was not excluded as the basis for that increase and could be of interest in the current context. The literature contains many examples of dehydrogenases (glucose and alcohol) requiring covalently or noncovalently bound quinonoids, termed quinoproteins (8), that are located in the periplasm or are peripherally associated with the outer surface of the cytoplasmic membrane as has been reported for AoxAB and AroAB, the As(III) oxidases thus far characterized.


arrow
ACKNOWLEDGMENTS
 
This work was primarily supported by the USDA-NRI Soils and Soil Biology Program (2002-35107-12268), with additional support provided by NASA Exobiology (NAG5-8807) and the Montana Agricultural Experiment Station (911310).

We also thank Terry Krulwich (Mount Sinai School of Medicine) for stimulating discussion regarding possible function of the Mrp antiporter.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717. Phone: (406) 994-2190. Fax: (406) 994-3933. E-mail: timmcder{at}montana.edu. Back


arrow
REFERENCES
 
    1
  1. Afkar, E., J. Lisak, C. Saltikov, P. Basu, R. S. Oremland, and J. F. Stolz. 2003. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10. FEMS Microbiol. Lett. 226:107-112.[CrossRef][Medline]
    2. 2
  2. Ahmann, D., A. L. Roberts, L. R. Krumholz, and F. M. M. Morel. 1994. Microbe grows by reducing arsenic. Nature 371:750.[CrossRef][Medline]
    3. 3
  3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. L. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
    4. 4
  4. Anderson, G. L., J. Williams, and R. Hille. 1992. The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase. J. Biol. Chem. 267:23674-23682.[Abstract/Free Full Text]
    5. 5
  5. Boos, W., and J. M. Lucht. 1996. Periplasmic binding protein-dependent ABC transporters, p. 1175-1209. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella. Cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.
    6. 6
  6. Cervantes, C., G. Ji, J. L. Ramirez, and S. Silver. 1994. Resistance to arsenic compounds in microorganisms. FEMS Microbiol. Rev. 15:355-367.[CrossRef][Medline]
    7. 7
  7. Cullen, W. R., and K. J. Reimer. 1989. Arsenic speciation in the environment. Chem. Rev. 89:713-764.[CrossRef]
    8. 8
  8. Diehl, A., F. von Wintzingerode, and H. Gorisch. 1998. Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer: sequence of the gene and deduced structural properties of the enzyme. Eur. J. Biochem. 257:409-419.[Medline]
    9. 9
  9. Ditta, G., T. Schmidhauser, E. Yakobson, P. Lu, X. Liang, D. R. Finlay, D. Guiney, and D. R. Helinski. 1985. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13:149-153.[CrossRef][Medline]
    10. 10
  10. Donahoe-Christiansen, J., C. R. Jackson, S. D'Imperio, W. P Inskeep, and T. R. McDermott. 2004. Arsenite-oxidizing Hydrogenobaculum sp. isolated from an acid-sulfate-chloride geothermal spring in Yellowstone National Park. Appl. Environ. Microbiol. 70:1865-1868.[Abstract/Free Full Text]
    11. 11
  11. Earhart, C. F. 1996. Uptake and metabolism of iron and molybdenum, p. 1075-1090. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella. Cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.
    12. 12
  12. Ellis, P. J., T. Conrads, R. Hille, and P. Kuhn. 2001. Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 Å and 2.03 Å. Structure 9:125-132.[Medline]
    13. 13
  13. Fearnley, I. M., and J. E. Walker. 1992. Conservation of sequences of subunits of mitochondrial complex I and their relationships with other proteins. Biochim. Biophys. Acta 1140:105-134.[Medline]
    14. 14
  14. Gihring, T. M., and J. F. Banfield. 2001. Arsenite oxidation and arsenate respiration by a new Thermus isolate. FEMS Microbiol. Lett. 204:335-340.[CrossRef][Medline]
    15. 15
  15. Gihring, T. M., G. K. Druschel, R. J. McCleskey, R. J. Hamers, and J. F. Banfield. 2001. Rapid arsenite oxidation by Thermus aquaticus and Thermus thermophilus: field and laboratory investigations. Environ. Sci. Technol. 35:3857-3862.[Medline]
    16. 16
  16. Green, H. H. 1918. Isolation and description of a bacterium causing oxidation of arsenite to arsenate in cattle dripping baths. Rep. Dir. Vet. S. Afr. 6:593-599.
    17. 17
  17. Hiramatsu, T., K. Kodama, T. Kuroda, T. Mizushima, and T. Tsuchiya. 1998. A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus. J. Bacteriol. 180:6642-6648.[Abstract/Free Full Text]
    18. 18
  18. Huber, R., M. Sacher, A. Vollmann, H. Huber, and D. Rose. 2000. Respiration of arsenate and selenate by hyperthermophilic archaea. Syst. Appl. Microbiol. 23:305-314.[Medline]
    19. 19
  19. Inskeep, W. P., T. R. McDermott, and S. E. Fendorf. 2001. Arsenic (V)(III) cycling in soils and natural waters: chemical and microbiological processes, p. 183-215. In W. F. Frankenberger, Jr., and J. M. Macy (ed.), Environmental chemistry of arsenic. Marcel Dekker, New York, N.Y.
    20. 20
  20. Ito, M., A. A. Guffanti, B. Oudega, and T. A. Krulwich. 1999. Mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis. J. Bacteriol. 181:2394-2402.[Abstract/Free Full Text]
    21. 21
  21. Ito, M., A. A. Guffanti, and T. A. Krulwich. 2001. Mrp-dependent Na+/H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters. FEBS Lett. 496:117-120.[CrossRef][Medline]
    22. 22
  22. Ji, G., S. Silver, E. A. E. Garber, H. Ohtake, C. Cervantes, and P. Corbisier. 1993. Bacterial molecular genetics and enzymatic transformations of arsenate, arsenite and chromate, p. 529-539. In A. E. Torma, M. L. Apel, and C. L. Brierley (ed.), Biohydrometallurgical technologies, vol. 2. The Minerals, Metals, & Materials Society, Warrendale, Pa.
    23. 23
  23. Kashyap, D. R., L. M. Botero, W. L. Franck, D. J. Hassett, and T. R. McDermott. 2006. Complex regulation of arsenite oxidation in Agrobacterium tumefaciens. J. Bacteriol. 188:1081-1088.[Abstract/Free Full Text]
    24. 24
  24. Klemperer, N. S., and C. M. Pickart. 1989. Arsenite inhibits two steps in the ubiquitin-dependent proteolytic pathway. J. Biol. Chem. 264:19245-19252.[Abstract/Free Full Text]
    25. 25
  25. Krafft, T., and J. M. Macy. 1998. Purification and characterization of the respiratory arsenate reductase of Chysiogenes arsenatis. Eur. J. Biochem. 255:647-653.[Medline]
    26. 26
  26. Krulwich, T. A., M. Ito, and A. A. Guffanti. 2001. The Na+-dependence of alkaliphily in Bacillus. Biochim. Biophys. Acta 1505:158-168.[Medline]
    27. 27
  27. Lett, M. C., K. Paknikar, and D. Lievremont. 2001. A simple and rapid method for arsenite and arsenate speciation. In V. S. T. Ciminelli and O. Garcia, Jr. (ed.), Biohydrometallurgy—fundamentals, technology and sustainable development, part B. Elsevier Science, New York, N.Y.
    28. 28
  28. Lopez-Corcuera, G., M. Bastidas, and M. Dubourdieu. 1993. Molybdenum uptake in Escherichia coli K-12. J. Gen. Microbiol. 139:1869-1875.[Medline]
    29. 29
  29. Macur, R. E., C. R. Jackson, T. R. McDermott, and W. P. Inskeep. 2004. Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil. Environ. Sci. Technol. 38:104-111.[Medline]
    30. 30
  30. Mathiesen, C., and C. Hagerhall. 2002. Transmembrane topology of the NuoL, M and N subunits of NADH:quinoneoxidoreductases and their homologues among membrane-bound hydrogenases and bona fide antiporters. Biochim. Biophys. Acta 1556:121-132.[Medline]
    31. 31
  31. Mathiesen, C., and C. Hagerhall. 2003. The "antiporter module" of respiratory chain complex I includes the MrpC/NuoK subunit—a revision of the modular evolution scheme. FEBS Lett. 5459:7-13.
    32. 32
  32. McDermott, T. R., and M. L. Kahn. 1992. Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene. J. Bacteriol. 174:4790-4797.[Abstract/Free Full Text]
    33. 33
  33. Meng, Y.-L., Z. Liu, and B. P. Rosen. 2005. As(III) and Sb(III) uptake by GlpF and efflux by ArsB in Escherichia coli. J. Biol. Chem. 279:18334-18341.
    34. 34
  34. Mukhopadhyay, R., B. P. Rosen, L. T. Phung, and S. Silver. 2002. Microbial arsenic: from geocycles to genes and enzymes. FEMS Microbiol. Rev. 26:311-325.[CrossRef][Medline]
    35. 35
  35. Muller, D., D. Lievremont, D. D. Simeonova, J.-C. Hubert, and M.-C. Lett. 2003. Arsenite oxidase aox genes from a metal-resistant ß-proteobacterium. J. Bacteriol. 185:135-141.[Abstract/Free Full Text]
    36. 36
  36. Norris, T. B., J. Wraith, R. C. Castenholz, and T. R. McDermott. 2002. Soil microbial community diversity after a recent geothermal heating event. Appl. Environ. Microbiol. 68:6300-6309.[Abstract/Free Full Text]
    37. 37
  37. Oremland, R. S., S. E. Hoft, J. M. Santini, N. Bano, R. A. Hollibaugh, and J. T. Hollibaugh. 2002. Anaerobic oxidation of arsenite in Mono Lake water by a facultative, arsenite-oxidizing chemoautotroph, strain MLHE-1. Appl. Environ. Microbiol. 68:4795-4802.[Abstract/Free Full Text]
    38. 38
  38. Oremland, R. S., and J. F. Stolz. 2003. The ecology of arsenic. Science 300:939-944.[Abstract/Free Full Text]
    39. 39
  39. Osborne, F. H., and H. L. Ehrlich. 1976. Oxidation of arsenite by a soil isolate of Alcaligenes. J. Appl. Bacteriol. 41:295-305.[Medline]
    40. 40
  40. Parvatiyar, K., E. Alsabbagh, U. A. Ochsner, M. A. Stegemeyer, A. G. Smulian, S. H. Hwang, C. R. Jackson, T. R. McDermott, and D. J. Hassett. 2005. Global analysis of cellular factors and responses involved in Pseudomonas aeruginosa resistance to arsenite. J. Bacteriol. 187:4853-4864.[Abstract/Free Full Text]
    41. 41
  41. Phillips, S. E., and M. L. Taylor. 1976. Oxidation of arsenite to arsenate by Alcaligenes faecalis. Appl. Environ. Microbiol. 32:392-399.[Abstract/Free Full Text]
    42. 42
  42. Pontius, F. W., K. G. Brown, and C.-J. Chen. 1994. Health implications of arsenic in drinking water. J. Am. Water Works Assoc. 86:52-63.
    43. 43
  43. Saier, M. H., Jr., B. H. Eng, S. Fard, J. Garg, D. A. Haggerty, W. J. Hutchinson, D. L. Jack, E. C. Lai, H. J. Liu, D. P. Nusinew, A. M. Omar, S. S. Pao, I. T. Paulsen, J. A. Quan, M. Sliwinski, T. T. Tseng, S. Wachi, and G. B. Young. 1999. Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422:1-56.[Medline]
    44. 44
  44. Salmassi, T. M., K. Venkateswaren, M. Satomi, K. H. Nealson, D. K. Newman, and J. G. Hering. 2002. Oxidation of arsenite by Agrobacterium albertimagni, AOL15, sp. nov., isolated from Hot Creek, California. Geomicrobiol. J. 19:53-66.
    45. 45
  45. Saltikov, C., and D. K. Newman. 2003. Genetic identification of a respiratory arsenate reductase. Proc. Natl. Acad. Sci. USA 100:10983-10988.[Abstract/Free Full Text]
    46. 46
  46. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    47. 47
  47. Santini, J. M., L. I. Sly, R. D. Schnagl, and J. M. Macy. 2000. A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies. Appl. Environ. Microbiol. 66:92-97.[Abstract/Free Full Text]
    48. 48
  48. Santini, J. M., and R. N. vanden Hoven. 2004. Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26. J. Bacteriol. 186:1614-1619.[Abstract/Free Full Text]
    49. 49
  49. Silver, S., and L. T. Phung. 2005. Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Appl. Environ. Microbiol. 71:599-608.[Free Full Text]
    50. 50
  50. Silver, S., L. T. Phung, and B. P. Rosen. 2002. Arsenic metabolism: resistance, reduction, and oxidation, p. 247-272. In W. F. Frankenberger, Jr., and J. M. Macy (ed.), Environmental chemistry of arsenic. Marcel Dekker, New York, N.Y.
    51. 51
  51. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784-791.[CrossRef]
    52. 52
  52. Somerville, J. E., and M. L. Kahn. 1983. Cloning of the glutamine synthetase I gene from Rhizobium meliloti. J. Bacteriol. 156:168-176.[Abstract/Free Full Text]
    53. 53
  53. Summers, M. L., J. G. Elkins, B. Elliot, and T. R. McDermott. 1998. Expression and regulation of phosphate starvation inducible genes in Rhizobium meliloti. Mol. Plant-Microbe Interact. 11:1094-1101.[Medline]
    54. 54
  54. Swartz, T. H., S. Ikewada, O. Ishikawa, M. Ito, and T. A. Krulwich. 2005. The Mrp Na+/H+ antiporter increases the activity of the malate:quinone oxidoreductase of an Escherichia coli respiratory mutant. J. Bacteriol. 187:388-391.[Abstract/Free Full Text]
    55. 55
  55. Swartz, T. H., M. Ito, D. B. Hicks, M. Nuqui, A. A. Guffanti, and T. A. Krulwich. 2005. The Mrp system: a giant among monovalent cation/proton antiporters? Extremophiles 9:345-354.[CrossRef][Medline]
    56. 56
  56. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
    57. 57
  57. Weeger, W., D. Lievremont, M. Peret, F. Lagarde, J. C. Hubert, M. Leroy, and M. C. Lett. 1999. Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment. Biometals 12:141-149.[CrossRef][Medline]

Journal of Bacteriology, February 2006, p. 1577-1584, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1577-1584.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kajiyama, Y., Otagiri, M., Sekiguchi, J., Kudo, T., Kosono, S. (2009). The MrpA, MrpB and MrpD subunits of the Mrp antiporter complex in Bacillus subtilis contain membrane-embedded and essential acidic residues. Microbiology 155: 2137-2147 [Abstract] [Full Text]  
  • Morino, M., Natsui, S., Swartz, T. H., Krulwich, T. A., Ito, M. (2008). Single Gene Deletions of mrpA to mrpG and mrpE Point Mutations Affect Activity of the Mrp Na+/H+ Antiporter of Alkaliphilic Bacillus and Formation of Hetero-Oligomeric Mrp Complexes. J. Bacteriol. 190: 4162-4172 [Abstract] [Full Text]  
  • D'Imperio, S., Lehr, C. R., Breary, M., McDermott, T. R. (2007). Autecology of an Arsenite Chemolithotroph: Sulfide Constraints on Function and Distribution in a Geothermal Spring. Appl. Environ. Microbiol. 73: 7067-7074 [Abstract] [Full Text]  
  • Swartz, T. H., Ito, M., Ohira, T., Natsui, S., Hicks, D. B., Krulwich, T. A. (2007). Catalytic Properties of Staphylococcus aureus and Bacillus Members of the Secondary Cation/Proton Antiporter-3 (Mrp) Family Are Revealed by an Optimized Assay in an Escherichia coli Host. J. Bacteriol. 189: 3081-3090 [Abstract] [Full Text]  
  • Lehr, C. R., Kashyap, D. R., McDermott, T. R. (2007). New Insights into Microbial Oxidation of Antimony and Arsenic. Appl. Environ. Microbiol. 73: 2386-2389 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kashyap, D. R.
Right arrow Articles by McDermott, T. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kashyap, D. R.
Right arrow Articles by McDermott, T. R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»