11
Bacterial
Respiration
of
Arsenate
and
Its
Significance
in
the
Environment
Ronald
S.
Oremland
U.S.
Geological
Survey,
Menlo
Park,
California
Dianne
K.
Newman
California
Institute
of
Technology,
Pasadena,
California
Brian
W.
Kail
and
Johr:-
F.
Stolz
Duquesne
University,
Pittsburgh,
Pennsylvania
I.
INTRODUCTION
Although
arsenic
is
a trace
element
in
terms
of
its
natural
abundance,
it
nonethe-
less
has
a common
presence
within
the
earth's
crust.
Because
it
is
classified
as
a
group
VB
element
in
the
periodic
table,
it
shares
many
chemical
and
biochemical
properties
in
common
with
its
neighbors
phosphorus
and
nitrogen.
Indeed,
in
the
case
of
this
element's
most
oxidized
(+5)
oxidation
state,
arsenate
[HAsO/-
or
As
(V)],
its
toxicity
is
based
on
its
action
as
an
analog
of
phosphate.
Hence,
arsenate
ions
uncouple
the
oxidative
phosphorylation
normally
associated
with
the
enzyme
glyceraldehyde
3-phosphate
dehydrogenase,
thereby
preventing
the
formation
ofphosphoglyceroyl
phosphate,
a key
high-energy
intermediate
in
gly-
colysis.
To
guard
against
this,
a number
of
bacteria
possess
a detoxifying
arsenate
reductase
pathway
(the
arsC
system)
whereby
cytoplasmic
enzymes
remove
in-
ternal
pools
of
arsenate
by
achieving
its
reduction
to
arsenite
[H
2
As0
3
-
or
As
(III)].
However,
because
the
arsenite
product
binds
with
internal
sulfhydryl
groups
that
render
it
even
more
toxic
than
the
original
arsenate,
efficient
arsenite
efflux
from
the
cell
is
also
required
and
is
achieved
by
an
active
ion
''pumping''
273
274
Oremland
et
al.
system
(I).
The
details
of
this
bacterial
arsenic
detoxification
phenomenon
have
been
well
established
in
the
literature,
and
Chapter
I 0
in
this
volume
provided
a thorough
review.
Here,
we
discuss
bacterial
respiration
of
arsenate
and
its
sig-
nificance
in
the
environment.
As
a biological
phenomenon.
respiratory
growth
on
arsenate
is quite
remarkable.
given
the
toxicity
of
the
element.
Moreover,
the
consequences
of
microbial
arsenate
respiration
may,
at
times,
have
a significant
impact
on
environmental
chemistry.
Much
less
is
understood
about
the
mechanisms
of
microbial
arsenate
respi-
ration
than
about
the
mechanisms
of
arsenate
detoxification,
although
these
pro-
cesses
face
similar
challenges,
namely:
the
transport
of
arsenate
into
the
cell,
its
reduction
to
arsenite.
the
protection
of
intracellular
proteins
from
arsenite,
and
the
export
of
arsenite
out
of
the
cell.
In
the
case
of
arsenate-respiring
micro-
organisms,
the
nature
of
arsenic
transport
across
the
cell
membrane
is
as
yet
unclear,
and
we
must
look
to
arsenic-resistant
organisms
as
our
initial
models.
Presumably,
for
gram-negative
bacteria.
arsenate
enters
the
cells
through
nonspe-
cific
outer-membrane
porins,
or,
in
the
case
of
phosphate
starvation,
through
in-
ducible
outer-membrane
proteins
that
arc
designed
for
phosphate
transport
but
cannot
discriminate
between
arsenate
and
phosphate
(such
as
PhoE)
(2).
Upon
reaching
the
periplasm,
arsenate
may
be
reduced
to
arsenite
by
respiratory
reduc-
tases
(see
below)
or
further
transported
into
the
cytoplasm,
as
is
known
to
occur
in
Esclzericlzia
coli
and
presumably
in
other
As-resistant
organisms.
Under
condi-
tions
of
phosphate
abundance
(>I
mM),
arsenate
can
enter
the
cytoplasm
through
a
low-afflnity
phosphate
transport
system
(Pit).
In
E.
coli,
pitA
is
constitutively
expressed
and
couples
uptake
of
inorganic
phosphate
to
the
proton
motive
force
(3
).
In
strains
dependent
on
Pit
for
inorganic
phosphate
uptake,
exposure
to
arse-
nate
leads
to
the
depletion
of
intracellular
adenosine
triphosphate
(ATP)
stores
and
the
intracellular
accumulation
of
arsenate,
demonstrating
the
direct
interfer-
ence
of
arsenate
in
phosphate
metabolism
(4).
Despite
the
similarity
between
arsenate
and
phosphate,
organisms
have
evolved
ways
to
discriminate
between
the
two
compounds.
For
example,
muta-
tions
in
pitA
lead
to
the
induction
of
the
high-affinity
phosphate
transport
system
(Pst),
which
differentiates
between
arsenate
and
phosphate
approximately
I 00-
fold
more
accurately
than
PitA.
The
key
components
of
the
Pst
system
include
PstS.
PstA,
PstB,
and
PstC.
PstS
is
a periplasmic
protein
that
hinds
inorganic
phosphate
with
high
selectivity
and
carries
it
to
the
high-affinity
phosphate trans-
porter
(located
in
the
cytoplasmic
membrane)
comprised
of
PstA.
PstC,
and
PstB.
Transport
of
phosphate
through
this
complex
is
coupled
to
A
TP
hydrolysis
at
PstB,
making
transport
of
phosphate
through
the
Pst
system
more
costly
for
the
cell
than
through
Pit
(2).
The
switch
from
the
low-
to
high-affinity
phosphate
transport
systems
results
in
moderate
arsenate
tolerance
(5).
Whether
arsenate-
respiring
organisms
also
rely
on
phosphate
transport
systems
to
uptake
arsenic
or
have
evolved
arsenate-specific
transporters
remains
an
open
question.
Bacterial
Respiration
of
Arsenate
275
Once
inside
the
cell,
arsenate
ions
can
be
reduced
to
arsenite
via
membrane-
bound
or
cytoplasmic
enzymes.
The
former
are
linked
to
cellular
energy
conser-
vation
and
are
described
in
detail
later
in
this
chapter
and
in
Chapter
12;
the
latter
are
characteristic
of
As-resistant
microbes,
do
not
conserve
energy,
and
have
been
described
in
Chapter
I
0.
Before
detailing
the
bioinorganic
chemistry
of
arsenic
by
micro-organisms,
however,
we
will
briefly
discuss
the
incorporation
of
arsenic
into
organic
compounds.
For
more details
on
this
subject,
we
refer
the
reader
to
comprehensive
reviews
by
Phillips
(6)
and
Reimer
(7).
Depending
upon
the
organism,
inorganic
arsenic
ultimately
may
be
con-
verted
into
arsenosugars
as
well
as
a variety
of
methylated
species.
The
occur-
rence
of
this
phenomenon
in
plants
and
algae
is
highly
variable.
Internal
arsenic
pools
occurring
in
mosses,
for
example,
are
dominated
by
inorganic
species
as
opposed
to
organic
compounds
(8).
Once
inside
the
cell,
however,
arsenate
may
undergo
further
reduction
to
a lower
redox
state
(arsine)
where
it
is
incorporated
into
organic
matter
in
a fashion
analogous
to
that
of
quaternary
nitrogen
com-
pounds.
For
example,
arsenobetaine
is
an
analog
of
the
internal
osmolyte
glycine
betaine
commonly
found
in
marine
organisms
(Fig.
I).
Arsenobetaine
commonly
occurs
in
a
·variety
of
marine
animals
(9),
as
well
as
in
bacteria
found
in
the
arsenic-rich
waters
of
hypersaline
Mono
Lake,
California
(I
0).
In
the
latter
case,
arsenobetaine
may
serve
a dual
purpose
of
functioning
not
only
as
a compatible
internal
solute
in
an
environment
of
high
osmotic
stress,
but
also
as
a mechanism
for
converting
potentially
toxic
internal
pools
of
arsenate
and
arsenite
into
an
innocuous
organic
compound.
Animals
presumably
obtain
their
organoarsenic
compounds
from
the
food
chain,
by
eating
As-containing
plants
and
algae.
The
occurrence
of
organoarsenicals
in
the
tissues
of
deep-sea
hydrothermal
vent
ani-
CH3
CH3-N(+)
-CHzCOo(-)
CH3
glycine
betaine
CH3
CH3-As(+)_CHzCOO(-)
CH3
arsenobetaine
Figure
1
Molec~
structure
of
the
osmolyte
glycine
betaine
and
its
analog
arseno-
betaine.
276
Oremland
et
al.
mals
suggests
that
it is also
possible for
animals
to
obtain
arsenic
from
autotrophic
and/or
symbiotic
bacterial
sources
rather
than
phytoplankton
(II).
Neff
(
12)
has
reviewed
the
toxicology
of
various
arsenic
species
in
marine
ecosystems.
A
number
of
micro-organisms
isolated
from
sediments,
macroalgae,
and
the
intestinal
tract
of
chitons
have
been
shown
to
degrade
arsenobetaine
to
tri-
methylarsine
oxide
[(CH1)1AsOI,
dimethylarsinic
acid
[(CH1hAsO(OH)],
meth-
ylarsonic
acid
ICH1As0(0H)
2
I,
arsenite,
and
arsenate.
In
this
pathway,
arsenic
is
oxidized
back
to
the
+
5 state
(9).
In
anoxic
environments,
glycine
betaine
is
cleaved
to
form
acetate
and
trimethylamine,
both
of
which
may
enter
methano-
genic
degradation
pathways
(
13).
Little
is
known
about
the
occurrence
of
trimeth-
ylarsine
in
nature,
although
methylated
arsenic
compounds
with
arsenic
in
the
+5
oxidation
state
have
been
detected
in
the
aerobic
regions
of
a number
of
water
bodies
(
14
),
presumably
from
aerobic
degradation
of
arsenobetaine
or
from
partial
oxidation
of
methyl
arsines.
Methanogenic
attack
of
trimethylarsine
would
result
in
the
formation
of
highly
toxic
arsine
gas
(AsH
1
)
rather
than
ammonia.
Relatively
little
is
known
about
the
biogeochemical
cycling
of
organoarsenic
compounds
in
the
aerobic
or
anaerobic
environments, making
this
an
area
ripe
for
future
investigation.
Although
the
arsC
system
of
bacterial
arsenate
resistance
and
the
decompo-
sition
of
organoarsenic
compounds
represent
potential
mechanisms
whereby
re-
duct•d
inorganic
arsenic
species
(arsenite
or
arsine)
can
accumulate
in
an
external
aqueous
milieu,
neither process
conserves
energy
or
offers
any
special
evolution-
ary
advantage
to
the
cells
other
than
survival
in
a toxic
aquatic
matrix.
It
was
the
report
by
Ahmann
et
al.
(
15)
in
1994
that
first
recognized
arsenate
as
an
anaerobic
terminal
electron
acceptor
capable
of
supporting
the
respiratory
growth
of
new
species
of
Eubactcria. The
discovery
of
this
phenomenon
has
implications
across
several
disciplines
of
environmental
importance,
including
microbiology,
biochemistry,
toxicology,
and
geochemistry,
and
is
the
subject
of
three
recent
succinct
reviews
(16-1
H).
Because
this
is a fast-emerging
field
and
the
phenome-
non
may
ultimately
impact
the
health
of
large
human
populations
in
such
regions
as
the
Ganges
delta
of
Banagladesh
and
India
(
19),
we
have
written
this
chapter
to
further
update
and
summarize
recent
findings.
II.
MICROBIOLOGY
AND
BIOCHEMISTRY
OF
ARSENATE
RESPIRATION
Currently,
in
pure
culture,
there
arc
seven
novel
species
of
Eubacteria,
most
of
them isolated
from
arsenic-rich
environments
(20-27),
that
are
capable
of
respir-
ing
arsenate
(Table
I).
Although
this
is
only
a small
number
of
representative
species,
it
is
already
clear
that
the
phenomenon
is
polyphyletic.
It occurs
in
both
gram-positive
(low
G
+
C)
Eubacteria
and
in
at
least
three
subdivisions
(delta,
Bacterial
Respiration
of
Arsenate
277
Table
1
Novel
Bacterial
and
Archaeal
Isolates
That
Can
Grow
by
Respiratory
Arsenate
Reduction
Microbe
Sulfurospirillum
arsenophilum
Sulfurospirillum
bamesii
Chrysiogenes
arsenatis
Desulfotomaculum
auripigmentum
Bacillus
arsenicoselenatis
Bacillus
selenitireducens
Desulfovibrio
Ben-RB
Pyrobaculum
arsenaticum
Classification
£-Proteobacteria
£-Proteobacteria
Deep-branch
Proteobacteria
Low
G
+
C
gram
positive
Low
G
+
C
gram
positive
Low
G
+
C
gram
positive
D-
Proteobacteria
Crenoarchaea
Refs.
15.
20
20,
21.
22
23
24,
25
26
26
27
28
epsilon,
and
gamma)
of
the
gram-negative
Proteobacteria,
along
with
another
deeply
branching
representative
(Chrysiogenes).
Preliminary
evidence
suggests
that
a marine strain
of
Shewanella
sp.
also
respires
arsenate
(D.
K.
Newman,
personal
communication).
Until
quite
recently
there
were
no
reports
of
Archaea
that
respire
arsenate,
although
the
elevated
concentrations
of
arsenic
commonly
occurring
in
hot
springs
and
in
some
hypersaline
lakes
suggests
a niche
in
which
arsenate-respiring
Crenoarchaea
and
Haloarchaea
patiently
await
discovery.
Re-
cently,
Huber
et
·a!.
(28)
reported
on
the
ability
of
a newly
isolated
hyperther-
mophilic
Crenoarchaea,
Pyrobaculum
arsenaticum,
to
respire
arsenate.
Examina-
tion
of
three
other
members
of
this
genus
revealed
that
one
of
them,
P.
aerophilum,
was
also
capable
of
arsenate
respiration.
Both
organisms
are
faculta-
tive
autotrophs,
being
able
to
grow
by
respiring
arsenate
with
H
2
as
the
electron
donor
and
C0
2
as
the
source
of
cell
carbon.
Two
organisms,
Bacillus
arsenicosel-
enatis
and
B.
selenitireducens
are
extremophiles
adapted
to
the
high
pH
and
salin-
ity
of
Mono
Lake,
California.
As
yet,
there
are
no
reports
of
extreme
acidophilic
Archaea
or
Bacteria
that
respire
arsenic.
However,
the
high
concentrations
of
arsenic
in
the
waters
of
Iron
Mountain,
California
(29)
and
the
occurrence
of
mats
of
iron-oxidizing
Archaea
in
this
system
(30)
would
suggest
the
presence
of
arsenic-oxidizing
and
-reducing
Archaea
as
well.
All
of
the
Eubacterial arsenate-respirers
reduce
arsenate
quantitatively
to
arsenite.
No
significant
gaseous
products
(methylated arsines
or
AsH
1
)
or
elemen-
tal
arsenic
are
produced.
None
of
the
arsenate
respirers
currently
in
culture
are
obligate
arsenate-reducers,
and
all
exhibit
various
degrees
of
flexibility
with
re-
gard
to
their
ability
to
use
electron
acceptors
other
than
arsenate.
For
example,
most
can
use
nitrate,
three
can
use
selenium
oxyanions,
two
are
microaerophiles,
and
two
are
sulfate-reducers.
Dissimilatory
nitrate
reduction
by
Sulfurospirillum
barnesii
results
in
the
formation
of
ammonium
rather
than
N
2
(21
).
Desulfotoma-
278
Oremland
et
al.
1.2
(a)
1.0
~
0.8
.s
*
0.6
c
Q)
~
0.4
<(
0.2
0
11.0
0.8
(b)
10.0
~
0.6
~
U>
c
.s
:::;;
a:
~
9.0
0.4
(1)
~
3
::;
~
U>
8.0
0.2
7.0
0
2
4
6
8
10
12°
Day
Figure
2
Sequential
reduction
of
(a)
arsenate
and
(h)
sulfate
during
growth
of
Desu(foto-
maculw11
auripigmentum.
(From
Ref.
24.)
cul11m
auripigment11m
respires
arsenate
first,
followed
by
sulfate
(Fig.
2),
re-
sulting
in
the
precipitation
of
yellow
arsenic
trisulfides
and
the
mineral
orpiment
(24.25).
The
precedence
of
As(V)
before
sulfate
can
be
explained
by
the
higher
energy
yield
(~G')
associated
with
the
oxidation
of
a given
electron
donor
(HJ
with
arsenate
(-23.03
kJ/mol
e )
as
opposed
to
sulfate
(-0.42
kJ/mol
e-)
(16).
However.
IJesu(fomicrohillm
strain
Ben-RB
exhibits
concurrent
reduction
of
ar-
senate
and
sulfate.
suggesting
that
there
arc
considerable
physiological
differ-
ences
that
factor
into
this
phenomenon
as
well
(27).
Growth
of
B.
arsenicoselenatis
on
lactate
results
in
its
oxidation
to
acetate
plus
C0
2
with
the
reduction
of
arsenate
to
arsenite
(Fig.
3
).
Growth
conforms
to
the
equation:
lactate
+ 2
HAsOc~
+ H
1
~acetate-+
2H
2
As01
+
HC01-
~G;'
=
-23.4
kJ/mole-
(I)
Bacterial
Respiration
of
Arsenate
279
12.0
109
10.0
~
8.0
1()8
.§.
d
c:
0
~
~
6.0
iii
c
~
CD
(,)
c:
0
4.0
107
(.)
2.0
0
24
36
lime
(h)
Figure
3
Growth
of
Bacillus
arsenicoselenatis
with
lactate
as
its
electron
donor
and
arsenate
as
its
electron
acceptor.
Symbols:
A,
arsenate;
/':,.,
arsenite;
•.
lactate;
D,
acetate;
e.
cells.
(From
Ref.
26.)
Therefore,
the
reduction
of
arsenate
is
highly
exergonic,
although
it
is not
nearly
as
potent
an
oxidant
as
are
selenate,
nitrate,
or
manganic
ions (
16).
To
date,
only
the
respiratory
arsenate
reductase
(DAsR)
from
C.
arsenatis
has
been
purified
and
characterized
(31
).
A
peri
plasmic
enzyme,
the
DAsR
is
composed
of
an
87-kDa
polypeptide
and
a 29-kDa
polypeptide.
It
is
a heterodimer
with
a native
molecular
mass
of
123
kDa.
The
active
site
is
located
in
the
large
subunit
and
contains molybdenum.
N-terminal
amino
acid
sequence
analysis
also
revealed
a putative
[Fe-S]
cluster binding
site.
The
small
subunit
contains
at
least
one
[Fe-S]
cluster
and
is believed
to
be
involved
in
electron
transfer
and
anchor-
ing.
A
small-molecular-mass
c-type
cytochrome
has
also
been
linked
to
the
en-
zyme complex.
The
enzyme
exhibits
a high
degree
of
specificity
for
arsenate
and
has
a
Km
of
300
J.LM.
How
arsenate
reduction
in
the
periplasm
is
coupled
to
the
generation
of
the
proton
motive
force
is
unclear,
however,
as
arsenate
reduction
should
consume
protons
and
electrons
(HAsO/-
+
4H+
+
2e-
~
HAs0
2
+
2H
2
0),
that
are
initially
generated from
the
oxidation
of
a substrate
like
hydrogen
(H
2
~
2
H+
+
2e-).
More
studies
are
needed
to
understand
the
details
of
the
electron
transfer
pathway
to
arsenate
in
this
organism,
and
we
refer
the
reader
to
Chapter
12
for
a more
comprehensive
discussion
of
this
system.