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New Arsenite-Oxidizing Bacteria Isolated from Australian Gold Mining
Environments--Phylogenetic Relationships
Joanne M. Santini; Lindsay I. Sly; Aimin Wen; Dean Comrie; Pascal De Wulf-Durand; Joan M. Macy
To cite this Article Santini, Joanne M. , Sly, Lindsay I. , Wen, Aimin , Comrie, Dean , De Wulf-Durand, Pascal and Macy,
Joan M.(2002) 'New Arsenite-Oxidizing Bacteria Isolated from Australian Gold Mining Environments--Phylogenetic
Relationships', Geomicrobiology Journal, 19: 1, 67 — 76
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New Arsenite-Oxidizing Bacteria
Isolated from Australian Gold Mining
Environments—Phylogenetic Relationships
JOANNE M. SANTINI
Downloaded By: [University College London] At: 12:08 11 August 2010
Department of Microbiology
La Trobe University
Melbourne, Victoria, Australia
LINDSAY I. SLY
AIMIN WEN
Centre for Bacterial Diversity and IdentiŽ cation
Department of Microbiology and Parasitology
University of Queensland
Brisbane, Queensland, Australia
DEAN COMRIE
Department of Microbiology
La Trobe University
Melbourne, Victoria, Australia
PASCAL DE WULF-DURAND
Centre for Bacterial Diversity and IdentiŽ cation
Department of Microbiology and Parasitology
University of Queensland
Brisbane, Queensland, Australia
JOAN M. MACY
Department of Microbiology
La Trobe University
Melbourne, Victoria, Australia
Received 18 May 2001; accepted 11 September 2001.
This work was supported by an Australian Research Council Grant (A09925054 ) and two Central Large La
Trobe University grants to JMM. We would like to thank D. Flood for technical assistance and M. Fegan, Cooperative Research Centre for Tropical Plant Pathology, University of Queensland , for assistance with phylogeneti c
analyses. We would also like to thank H. L. Ehrlich for the strain “Alcaligenes faecalis” HLE.
Address correspondenc e to Joanne M. Santini. E-mail: j.santini@latrobe.edu.au
67
68
J. M. Santini et al.
Nine novel arsenite-oxidizing bacteria have been isolated from two different gold mine
environments in Australia. Four of these organisms grow chemolithoautotrophically with
oxygen as the terminal electron acceptor, arsenite as the electron donor, and carbon
dioxide-bicarbonate as the sole carbon source. Five heterotrophic arsenite-oxidizing
bacteria were also isolated, one of which was found to be both phylogenetically and
physiologically identical to the previously described heterotrophic arsenite oxidizer
misidentiŽ ed as Alcaligenes faecalis. The results showed that this strain belongs to the
genus Achromobacter. Phylogenetically, the arsenite-oxidizing bacteria fall within two
separate subdivisions of the Proteobacteria. Interestingly, the chemolithoautotrophic
arsenite oxidizers belong to the ®-Proteobacteria , whereas the heterotrophic arsenite
oxidizers belong to the ¯-Proteobacteria.
Keywords
arsenite oxidation, chemolithoautotroph, phylogeny, gold mines
Downloaded By: [University College London] At: 12:08 11 August 2010
Introduction
Soluble forms of arsenic, such as arsenite [As(III); H3 AsO3 ] and arsenate [As(V); H2 AsO¡
4C
HC ] are frequently found in association with gold mines where the level of arsenopyrite
(FeAsS) is high (Tamaki and Frankenberger 1992; Wilkie and Hering 1998). Both arsenate
and arsenite are toxic to life; however, arsenite is considered the more toxic of these two
forms (Tamaki and Frankenberger 1992).
Chemical oxidation of arsenite to arsenate is very slow, yet microbial oxidation is rapid.
In environments where signiŽ cant amounts of arsenite are oxidized to arsenate within a short
period of time, this oxidation can be attributed to arsenite-oxidizing bacteria (Tamaki and
Frankenberger 1992).
Most of the previously described arsenite-oxidizing bacteria have been heterotrophic,
and the most common isolate has been Alcaligenes faecalis (Osborne and Ehrlich 1976;
Phillips and Taylor 1976; Ehrlich 1996). Only two organisms have been isolated that oxidize arsenite chemolithoautotrophically, using oxygen as the terminal electron acceptor,
arsenite as the electron donor, and carbon dioxide-bicarbonate as the sole carbon source.
The Ž rst of these organisms, Pseudomonas arsenitoxidans, was described in 1981 and has a
generation time in the order of 48 h (Ilyaletdinov and Abdrashitova 1981). More recently, a
Gram-negative, motile, rod-shaped bacterium, designated NT-26, was isolated from the
Granites gold mine, Northern Territory, Australia (Santini et al. 2000). This organism
is the fastest arsenite oxidizer ever reported with a doubling time of 7.6 h when grown
chemolithoautotrophically (Santini et al. 2000).
This study involved determining the different types of arsenite-oxidizing bacteria
present in two different arsenopyrite-containing gold mining environments in Australia,
the Central Deborah gold mine, Bendigo, Victoria, and the Granites gold mine, Northern
Territory. The results revealed that all but one of the nine arsenite-oxidizing bacteria isolated
were different to those previously identiŽ ed.
Material and Methods
Growth and Media Conditions
The minimal salts enrichment medium and the minimal salts growth medium used were
identical to those previously described for the isolation of NT-25 and NT-26 (Santini
et al. 2000). The concentrations of the electron donor, arsenite, included in the medium were 5 and 10 mM. The pH of both media was 8. All incubations were done at
28± C.
Arsenite-Oxidizing Bacteria
69
Source of Inocula
All samples were subterranean. The sample from Bendigo, Victoria, was arseniccontaminated water taken from 200 m below the Central Deborah gold mine (1.8 mg/l
arsenic). The samples from the Northern Territory were moist arsenopyrite-containing rock
taken from a mine tunnel approximately 300 m below the Granites gold mine. The samples
were placed into either 11 bottles (Bendigo samples) or minimal enrichment medium with
no arsenite (10 mL) (Northern Territory samples) and transported back to the laboratory
at ambient temperature. The water from Bendigo was used immediately (approximately
6 h after collection) for the enrichments. The samples from the Northern Territory were
incubated for a total of 7 days.
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Isolation of Arsenite-Oxidizers
The Bendigo water was inoculated into minimal salts enrichment medium containing
10 mM arsenite. The Northern Territory enrichment was subcultured into minimal salts
enrichment medium containing 10 mM arsenite. Once turbidity was detected in the enrichments they were subcultured several times into the same medium. Two different methods
were used to isolate the arsenite-oxidizing bacteria from the various enrichments: (1) Each
enrichment was serially diluted and spread onto minimal salts enrichment agar [2% (w/v)]
medium containing arsenite (10 mM). (2) The enrichment culture was streaked directly
onto minimal salts enrichment agar [2% (w/v)] medium containing arsenite (10 mM). After
growth, a number of different colonies were selected, puriŽ ed, and then tested for their
ability to grow in the minimal salts enrichment medium containing arsenite (10 mM).
To demonstrate that arsenite was oxidized to arsenate during growth, the isolates were
grown in minimal salts growth medium containing 5 mM arsenite upon which samples were
taken and analyzed for arsenite and arsenate (see Analytical Methods). The pH was also
measured, as arsenite oxidation results in a decrease in pH (Santini et al. 2000).
For example, when NT-26 was grown in minimal salts growth medium containing
5 mM arsenite, complete oxidation of arsenite resulted in a decrease in pH from 8.0 to 6.5
(Santini et al. 2000).
Analytical Methods
Arsenite was determined using a Varian Spectra AA20 atomic absorption spectrophotometer, with a VGA 76 hydride generator (Macy et al. 1996). Arsenate was determined using
high-pressure liquid chromatography (HPLC) (Macy et al. 1996) and inductively coupled
plasma (ICP) (Jobin Yvon 24; France).
PCR AmpliŽ cation of 16S rRNA Genes
A bacterial suspension (108 CFU/ml) of each isolate was boiled for 10 min to release the
DNA and centrifuged for 5 min in a microcentrifuge. The supernatant was used as the DNA
template for PCR ampliŽ cation of the 16S rRNA genes. PCR ampliŽ cation was performed
in a 100-¹l reaction volume containing PCR buffer [67 mM Tris-HCl (pH 8.8), 16.6 mM
(NH4 )2 SO4 , 0.45% (v/v) Triton X-100, 200 ¹g ml¡1 of gelatin] (Biotech International, Ltd.,
Perth, Australia), 1.5 mM MgCl2 , each deoxynucleotide phosphate at a concentration of 200
¹M, 0.25 ¹M primer 27f (Lane 1991), 0.25 ¹M primer 1525r (Lane 1991), 5 ¹l of lysed
cells, and 2 U of Tth Plus DNA polymerase (Biotech International Ltd., Perth, Australia).
All PCR ampliŽ cations were performed in a Perkin-Elmer Cetus model 480 thermal cycler
(Applied Biosystems, Foster City, California, USA). The PCR conditions consisted of an
70
J. M. Santini et al.
initial denaturation step at 96± C for 5 min, 28 cycles of 48± C for 1 min, 72± C for 2 min,
94± C for 1 min, and one additional cycle at 48± C for 1 min and 72± C for 5 min to allow all
extension products to be completed. The PCR products were puriŽ ed by using the Promega
Wizard Minipreps DNA puriŽ cation system according to the manufacturer’s instructions
(Promega Corporation, Annandale, NSW, Australia).
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16S rDNA Sequencing
The puriŽ ed PCR product was used as the template for sequencing. A Taq DyeDeoxy TM
Terminator Cycle sequencing kit (Applied Biosystems, Foster City, California, USA) or
the ABI PRISMTM Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster
City, California, USA) was used following procedures recommended by the manufacturer.
The following nine 16S rDNA sequencing primers were used in the sequencing reactions:
27f, 342r, 357f, 519r, 530f, 907r, 1114f, and 1525r (Lane 1991) and 803f (Stackebrandt
and Charfreitag 1990). The sequencing products were puriŽ ed according to the manufacturer’s instructions. The sequences were determined on an Applied Biosystems 373A DNA
sequencer.
Phylogenetic Analysis
The near full-length 16S rDNA sequences determined were aligned with the sequences of
Escherichia coli and reference sequences of members of the ®- or ¯-Proteobacteria using
the Fast Aligner (V1.02) within the ARB EDIT tool of the ARB software for the analysis
of sequence data [Department of Microbiology, Technische Universität München, Munich,
Germany (http://www.mikro.biologie.tu-muenchen.de)]. The alignment was checked by eye
and some corrections were made manually. Evolutionary similarities and distances were
calculated using the Felsenstein (1993) correction, and a phylogenetic tree was constructed
using the neighbor-joining method of Saitou and Nei (1987). Bootstrap analysis of 100 data
resamplings was performed with SEQBOOT and CONSENSE (Felsenstein 1993) to determine the statistical conŽ dence of branch points in the tree.
The following sequences of bacterial strains with strain numbers where available
were obtained from GenBank and the Ribosomal Database Project (RDP) (Maidak et al.
1997) for inclusion in the phylogenetic analyses: Acidovorax avenae subsp. avenae ATCC
19860 (accession no. AF078759), Achromobacter piechaudii ATCC 43552 (accession no.
AB010841 ), Achromobacter ruhlandii ATCC 15749 (accession no. AB010840), Achromobacter xylosoxydans subsp. denitriŽ cans ATCC 15173 (accession no. M22509), Achromobacter xylosoxydans subsp. xylosoxydans ATCC 27061 (accession no. D88005),
Achromobacter sp. strain 3– 17 (accession no. U80417), “Acinetobacter” sp. strain IF-19
(accession no. X86602), Agrobacterium rhizogenes IFO 13257 (accession no. D14501),
Agrobacterium rubi LMG 156 (accession no. X67228), Agrobacterium tumefaciens NCPPB
2437 (accession no. D14500), Agrobacterium vitis NCPPB 3354 (accession no. D14502),
Alcaligenes faecalis ATCC 8750 (accession no. M225081), Aquaspirillum gracile ATCC
19624 (accession no. AF078753), Aquaspirillum psychrophilum LMG 5408 (accession no.
AF078755), Aquaspirillum sinuosum LMG 4393 (accession no. AF078754), Blastobacter
aggregatus (accession no. X73041), Blastobacter capsulatus (accession no. X73042), Bordetella avium ATCC 35086 (accession no. U04947), Bordetella bronchiseptica ATCC 19395
(accession no. U04948), Bordetella holmesii CDC F101 (accession no. U04820), Bordetella
parapertussis ATCC 15311 (accession no. U04949), Bordetella pertussis ATCC 9797 (accession no. U04950), Brachymonas denitriŽ cans strain AS-P1 (accession no. D14320),
Burkholderia caryophylli ATCC 25418 (accession no. X67039), Burkholderia cepacia
ATCC 25416 (accession no. M22518), Comamonas testosteroni ATCC 11996 (accession
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Arsenite-Oxidizing Bacteria
71
no. M11224), Delftia acidovorans strain ACM 489 (accession no. AF078774), Herbaspirillum seropedicae (accession no. Y10164), Hydrogenophag a  ava CCUG 1658 (accession no. AF078771), Hydrogenophaga palleronii CCUG 20334 (accession no. AF078769),
Hydrogenophag a pseudo ava ATCC 33668 (accession no. AF078770), Hydrogenophaga
sp. DSM 5680 (accession no. AF019037), Hydrogenophaga taeniospiralis ATCC 49743
(accession no. AF078768), Ideonella dechloratans (accession no. X72724), Leptothrix
discophora ATCC 43182 (accession no. L33975), Mesorhizobium huakuii IFO 15243
(accession no. D13431), Mesorhizobium loti LMG 6125 (accession no. X67229), Mesorhizobium mediterraneum UPM-Ca36 (accession no. L38825), Oxalobacter formigenes ATCC
35274 (accession no. ARB 776391B7 ), Polaromonas vacuolata strain 34 P (accession
no. U14585), Rhizobium etli CFN 42 (accession no. U28916), Rhizobium galegae ATCC
43677 (accession no. D11343), Rhizobium gallicum R602 (accession no. U86343), Rhizobium giardinii H152 (accession no. U86344), Rhizobium huautlense S02 (accession no.
AF025852 ), Rhizobium leguminosarum DSM 30132 (accession no. ARB 8B13E90D),
Rhizobium mongolense USDA 1844 (accession no. U89817), Rhizobium tropici USDA
9030 (accession no. U89832), Rhodoferax fermentans strain FR2 (accession no. D16211),
Rubrivivax gelatinosus strain ATH 2.2.1 (accession no. D16213), Sinorhizobium fredii
ATCC 35423 (accession no. D14516), Sinorhizobium medicae strain A321 (accession
no. L39882), Sinorhizobium meliloti LMG 6133 (accession no. X67222), Sinorhizobium
saheli LMG 7837 (accession no. X68390), Sinorhizobium terangae LMG 6463, (accession
no. X68387), Sinorhizobium xinjiangensis IAM 14142 (accession no. D12796), Telluria
chitinolytica ACM 3522 (accession no. X65590), Telluria mixta ACM 1762 (accession no.
X65589), Thiomonas cuprina DSM 5495 (accession no. U67162), arsenite-oxidizing bacterium strain NT-25 (accession no. AF159452), arsenite-oxidizing bacterium strain NT-26
(accession no. AF159453), Variovorax paradoxus IAM 12373 (accession no. D30793), and
Xylophilus ampelinis ATCC 33914 (accession no. AF078758).
Nucleotide Sequence Accession Numbers
The sequences determined in this study for strains NT-14, NT-5, NT-6, NT-10, NT-2, NT-3,
NT-4, BEN-4, BEN-5, and “Alcaligenes faecalis” strain HLE have been deposited in GenBank under the accession numbers AY027497 to AY027506, respectively.
Results
Isolation of Arsenite-Oxidizing Bacteria
The different arsenite-oxidizing bacteria isolated are listed in Table 1. For comparison
strains, NT-25 and NT-26 (Santini et al. 2000) and one of the original A. faecalis strains
(Osborne and Ehrlich 1976; Ehrlich 1996) [designated here as “A. faecalis” HLE to distinguish it from A. faecalis that does not oxidize arsenite] are included.
As can be seen, of the nine newly isolated arsenite-oxidizing bacteria, four grew
chemolithoautotrophically, all of which are members of the ®-Proteobacteria (see Phylogenetic Analysis of New Arsenite-Oxidizing Bacteria). The time required for these bacteria
to oxidize 5 mM arsenite in a deŽ ned growth medium, however, varied from 4 to 5 days
(for NT-2, NT-3, NT-4) to greater than 5 days (for BEN-5). For these bacteria, growth does
not occur in the absence of arsenite (i.e., only in an aerobic minimal salts medium containing carbon dioxide-bicarbonate) (data not shown). Interestingly, the Ž ve heterotrophic
arsenite-oxidizing bacteria isolated as part of this study and “A. faecalis” HLE are members
of the ¯-Proteobacteria (see next section). These organisms only oxidize arsenite when
grown in the presence of organic matter (e.g., yeast extract). All of the organisms isolated
72
J. M. Santini et al.
TABLE 1 Comparisons of the chemolithoautotrophic arsenite-oxidizing ability of
various newly isolated arsenite-oxidizing bacteria with “A. faecalis” HLE
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Bacteriuma
NT-2
NT-3
NT-4
NT-5
NT-6
NT-14
NT-10
NT-25
NT-26
BEN-4
BEN-5
“A. faecalis”
HLE
Chemolithoautotrophic
growth
C
C
C
¡
¡
¡
¡
C
C
¡
C
¡
Time required
(days) for complete
chemolithoautotrophic
oxidation of 5 mM arseniteb
Subgroup of the
Proteobacteria
4– 5
4– 5
4– 5
NA
NA
NA
NA
3
3
NA
>5
NA
®
®
®
¯
¯
¯
¯
®
®
¯
®
¯
a
“BEN” D isolated from the Central Deborah Mine in Bendigo; “NT” D isolated from the Granites
Gold Mine in the Northern Territory.
b
Aerobic growth in a minimal salts medium containing 5 mM arsenite as the electron donor and
carbon dioxide-bicarbonate as the sole carbon source.
NA D not applicable as these organisms require organic matter for growth and arsenite oxidation.
oxidized 5 mM arsenite completely to arsenate and in all cases the pH decreased from 8.0
to 6.5, which is indicative of arsenite oxidation.
Phylogenetic Analysis of New Arsenite-Oxidizing Bacteria
The phylogenetic analysis of almost complete 16S rDNA sequences (1,390 – 1,481 nucleotides) of the arsenite-oxidizing bacteria showed that they were phylogenetically dispersed in either the ®-Proteobacteria (Figure 1) or the ¯-Proteobacteria (Figure 2).
The analysis of 1,332 unambiguous nucleotide positions of those strains belonging
to the ®-Proteobacteria showed that strain BEN-5 isolated from Bendigo, Victoria, was
most closely related to Agrobacterium vitis (97.7% sequence similarity). The relationship
was conŽ rmed by Biolog phenotypic analysis, which showed that strain BEN-5 exhibited
0.407 similarity with A. vitis but at this level (<0.5) could not be deŽ nitely identiŽ ed as
belonging to this species. It is probable that strain BEN-5 belongs to a new species in the
genus Agrobacterium. The isolates NT-2, NT-3, and NT-4 from the Northern Territory exhibited 99.4% sequence similarity and belonged to a well-supported novel branch (100%
bootstrap value) within the genus Sinorhizobium. The three strains were most closely related to Sinorhizobium fredii and Sinorhizobium xinjiangensis (sequence similarity 99.3%),
however, because of the high sequence similarities with species in the genus, DNA-DNA
hybridization will be required to elucidate their species identity. The strains NT-2, NT-3,
and NT-4 were phylogenetically unrelated to strains NT-25 and NT-26 previously reported
(Santini et al. 2000) [6] to belong to the Agrobacterium-Rhizobium subbranch. These latter
two strains that share 99.8% sequence similarity are members of a cluster with moderate bootstrap support (76%) having closest sequence similarities to Rhizobium huautlense
(96.2%), Rhizobium galegae (96.6%), and to a misidentiŽ ed “Acinetobacter” sp. strain
73
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Arsenite-Oxidizing Bacteria
FIGURE 1 Neighbor-joining tree showing the phylogenetic relationship of arseniteoxidizing strains BEN-5, NT-2, NT-3, NT-4, NT-25, and NT-26 with species belonging
to the ®-Proteobacteria. The sequence of Mesorhizobium loti was used as the outgroup.
The analysis included data from 1,332 unambiguous nucleotide positions. SigniŽ cant bootstrap values from 100 analyses are shown at the branch points of the trees. The scale bar
represents 1 nucleotide substitution per 100 nucleotides of 16S rRNA sequence.
IF-19 (97.4%) isolated from a deep subsurface mine gallery (Boivin-Jahns et al. 1995).
It is likely that strains NT-25 and NT-26 belong to a new species of Rhizobium. However, as the sequence similarity to known species of Rhizobium is around 97%, DNA-DNA
hybridization and phenotypic characterization will be required to conŽ rm their separate
species status (Stackebrandt and Goebel 1994). The ability of these organisms to oxidize
arsenite to arsenate constitutes a novel feature that has not been previously described in the
J. M. Santini et al.
Downloaded By: [University College London] At: 12:08 11 August 2010
74
FIGURE 2 Neighbor-joining tree showing the phylogenetic relationship of arseniteoxidizing strains BEN-4, NT-5, NT-6, NT-10, and NT-14 with species belonging to the
¯-Proteobacteria. The sequence of Telluria mixta was used as the outgroup. The analysis
included data from 1,331 unambiguous nucleotide positions. SigniŽ cant bootstrap values
from 100 analyses are shown at the branch points of the trees. The scale bar represents
1 nucleotide substitution per 100 nucleotides of 16S rRNA sequence.
genera Agrobacterium, Sinorhizobium, and Rhizobium. These organisms are therefore the
Ž rst examples of these genera that are able to use arsenite oxidation for growth.
The arsenite-oxidizing strains belonging to the ¯ -Proteobacteria have three different
phylogenetic afŽ liations. Strains NT-5, NT-6, and NT-14 from the Northern Territory belong
to a strongly supported branch (99% bootstrap value) closely related to species of the genus
Hydrogenophaga. The three strains have 99.7% sequence similarity and share 97.1 to 98%
similarity with existing members of the genus Hydrogenophaga.
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Arsenite-Oxidizing Bacteria
75
Strain NT-10 belongs to a strongly supported lineage (100% bootstrap value) that
includes the genera Bordetella and Achromobacter. The low bootstrap support for the NT-10
branch makes it impossible to be more speciŽ c about its generic afŽ liation. The separation of
Bordetella avium from the main Bordetella cluster affects the ability to assign this organism
to either Achromobacter or Bordetella. Arsenite oxidation has not been previously reported
for members of the genus Bordetella, although it has been described in strains currently
assigned to the genus Achromobacter.
Strain BEN-4 belongs to the Achromobacter lineage. This lineage contains species that
have had an uncertain taxonomy and have in the past been assigned to the genera Alcaligenes
and Achromobacter. Strain BEN-4 is phylogenetically most closely related to Ehrlich’s
arsenite-oxidizing strain “A. faecalis” HLE previously misidentiŽ ed as Alcaligenes faecalis (Osborne and Ehrlich 1976). The close relationship indicated by a sequence similarity
of 99.8% between the two strains is supported by their common physiology. Phenotypically strain BEN-4 and strain HLE identify by Biolog as [Alcaligenes] xylosoxydans subsp.
denitriŽ cans or Alcaligenes piechaudii which could not be differentiated by Biolog. The
species [Alcaligenes] denitriŽ cans and [Alcaligenes] piechaudii have recently been reassigned to the genus Achromobacter as Achromobacter xylosoxidans subsp. denitriŽ cans
and Achromobacter piechaudii, respectively, by Yabuuchi and coworkers (1998) in a phylogenetic study that should provide future taxonomic stability to this group of bacteria.
Therefore, based on phylogenetic and phenotypic grounds both strains BEN-4 and
“A. faecalis” HLE belong to the genus Achromobacter, however, assignment to the species
level will require DNA-DNA hybridization studies due to the high 16S rRNA sequence
similarities between species of this genus.
Discussion
With the exceptions of “Pseudomonas arsenitoxidans” (Ilyaletdinov and Abdrashitova
1981) and NT-25/NT-26 (Santini et al. 2000), all of the previously described arsenite oxidizers were heterotrophic. Most were strains of A. faecalis (Osborne and Ehrlich 1976;
Phillips and Taylor 1976; Tamaki and Frankenberger 1992; Ehrlich 1996).
The discovery of four new chemolithoautotrophic arsenite-oxidizing bacteria isolated
from gold mines in different locations in Australia clearly demonstrates that energy for
growth can be conserved during the oxidation of arsenite to arsenate. Interestingly, all of
these organisms fall within two separate lineages of the ®-Proteobacteria—(1) NT-2/NT3/NT-4 in the Sinorrhizobium lineage and (2) BEN-5 in the Agrobacterium/Rhizobium
lineage to which the previously identiŽ ed NT-25 and NT-26 belong (Santini et al. 2000).
To date, arsenite oxidation has not been described in members of these genera. The rate
of chemolithoautotrophic arsenite oxidation, however, varies (Table 1). The fastest arsenite
oxidizer ever reported, NT-26, has been studied in detail and has a doubling time of 7.6 h
when grown chemolithoautotrophically with arsenite as the electron donor (Santini et al.
2000).
Five heterotrophic arsenite-oxidizing bacteria were also isolated from gold mine
environments in Australia. These organisms fall within two separate lineages of the
¯ -Proteobacteria, (1) NT-5/NT-6/NT-14 in the Hydrogenophaga lineage and (2) NT-10/BEN4 in the Bordetella-Achromobacter lineages. Within the latter lineage, BEN-4 was found
to be phylogenetically and phenotypically identical to the previously characterized heterotrophic arsenite oxidizer “A. faecalis” HLE. All of these organisms require organic matter
for growth and arsenite oxidation. The new isolates oxidize arsenite throughout logarithmic
growth (Santini and Macy, unpublished data). Because of the requirement of “A. faecalis”
HLE for organic matter, arsenite oxidation is considered to be part of a detoxiŽ cation
76
J. M. Santini et al.
mechanism rather than one that supports growth. Whether this is also the case for the
heterotrophic arsenite-oxidizing bacteria described in this study remains to be determined.
Interestingly, the organisms isolated from Bendigo were different from those isolated
from the Northern Territory. The reason for this is unknown, however, the Granites gold mine
is located in the middle of the central Australian desert, which contains little organic matter.
On the other hand, the Central Deborah mine is located directly under the city of Bendigo,
and surface water that ultimately passes into the gold mine tunnels contains some organic
matter. This difference may explain why strains similar to “A. faecalis” HLE were not
isolated from the Northern Territory. It may also explain why more chemolithoautotrophic
arsenite oxidizers were found in the Northern Territory compared to only one found in
Bendigo.
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