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Journal
of
Biotechnology
214
(2015)
171–181
Contents lists available at ScienceDirect
Journal
of
Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
DNA
microarray
analysis
of
the
cyanotroph
Pseudomonas
pseudoalcaligenes
CECT5344
in
response
to
nitrogen
starvation,
cyanide
and
a
jewelry
wastewater
V.M.
Luque-Almagroa,1,
M.P.
Escribanoa,1,
I.
Mansoa,
L.P.
Sáeza,
P.
Cabellob,
C.
Moreno-Viviána,
M.D.
Roldána,∗
aDepartamento
de
Bioquímica
y
Biología
Molecular,
Edificio
Severo
Ochoa,
1aPlanta,
Campus
de
Rabanales,
Universidad
de
Córdoba,
14071
Córdoba,
Spain
bDepartamento
de
Botánica,
Ecología
y
Fisiología
Vegetal,
Edificio
Celestino
Mutis,
Campus
de
Rabanales,
Universidad
de
Córdoba,
14071
Córdoba,
Spain
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
10
July
2015
Received
in
revised
form
18
September
2015
Accepted
25
September
2015
Available
online
30
September
2015
Keywords:
Cyanide
degradation
Jewelry
wastewater
Nitrogen
starvation
Pseudomonas
Microarrays
a
b
s
t
r
a
c
t
Pseudomonas
pseudoalcaligenes
CECT5344
is
an
alkaliphilic
bacterium
that
can
use
cyanide
as
nitrogen
source
for
growth,
becoming
a
suitable
candidate
to
be
applied
in
biological
treatment
of
cyanide-
containing
wastewaters.
The
assessment
of
the
whole
genome
sequence
of
the
strain
CECT5344
has
allowed
the
generation
of
DNA
microarrays
to
analyze
the
response
to
different
nitrogen
sources.
The
mRNA
of
P.
pseudoalcaligenes
CECT5344
cells
grown
under
nitrogen
limiting
conditions
showed
consid-
erable
changes
when
compared
against
the
transcripts
from
cells
grown
with
ammonium;
up-regulated
genes
were,
among
others,
the
glnK
gene
encoding
the
nitrogen
regulatory
protein
PII,
the
two-component
ntrBC
system
involved
in
global
nitrogen
regulation,
and
the
ammonium
transporter-encoding
amtB
gene.
The
protein
coding
transcripts
of
P.
pseudoalcaligenes
CECT5344
cells
grown
with
sodium
cyanide
or
an
industrial
jewelry
wastewater
that
contains
high
concentration
of
cyanide
and
metals
like
iron,
copper
and
zinc,
were
also
compared
against
the
transcripts
of
cells
grown
with
ammonium
as
nitrogen
source.
This
analysis
revealed
the
induction
by
cyanide
and
the
cyanide-rich
wastewater
of
four
nitrilase-
encoding
genes,
including
the
nitC
gene
that
is
essential
for
cyanide
assimilation,
the
cyanase
cynS
gene
involved
in
cyanate
assimilation,
the
cioAB
genes
required
for
the
cyanide-insensitive
respiration,
and
the
ahpC
gene
coding
for
an
alkyl-hydroperoxide
reductase
that
could
be
related
with
iron
homeostasis
and
oxidative
stress.
The
nitC
and
cynS
genes
were
also
induced
in
cells
grown
under
nitrogen
starvation
conditions.
In
cells
grown
with
the
jewelry
wastewater,
a
malate
quinone:oxidoreductase
mqoB
gene
and
several
genes
coding
for
metal
extrusion
systems
were
specifically
induced.
©
2015
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Large
amounts
of
wastewaters
with
cyanide
are
produced
by
industrial
activities
like
mining
and
metal
processing,
electroplat-
ing,
goal
coking
and
nitrile
polymers
synthesis
(Kumar
et
al.,
2011).
Mining
uses
cyanide
for
extracting
gold,
silver
and
other
metals
from
ores,
processes
that
are
considered
the
largest
producers
of
cyanide-containing
wastewaters.
These
liquid
residues
also
contain
heavy
metals
and
metalloids
that
increase
their
toxicity.
In
these
industrial
wastes
cyanide
can
be
found
not
only
as
free
ion
(CN−)
∗Corresponding
author.
E-mail
address:
bb2rorum@uco.es
(M.D.
Roldán).
1These
authors
have
contributed
equally
to
this
work.
but
also
as
metal–cyanide
complexes,
since
cyanide
strongly
binds
to
metals.
For
this
reason,
cyanide
is
toxic
for
most
living
organisms,
inhibiting
essential
metalloenzymes
like
the
cytochrome
c
oxidase,
thus
blocking
aerobic
respiration
(Jünemann,
1997;
Quesada
et
al.,
2007).
The
jewelry
industry
also
produces
large
amounts
of
a
liquid
waste
that
contains
high
concentrations
of
cyanide
and
metals.
Thus,
the
residue
generated
by
the
jewelry
industry
of
Córdoba
(Spain)
displays
an
extremely
alkaline
pH
(>13)
and
contains
about
40
g
l−1cyanide
(ca.
1.5
M)
and
several
metals
like
copper,
iron
and
zinc.
Metals
are
required
at
very
low
concentrations
by
microor-
ganisms,
but
metal
accumulation
at
high
concentrations
cause
cell
toxicity,
which
is
mainly
related
with
their
capacity
to
interact
with
cellular
thiol
components
such
as
glutathione
(Nies,
2003).
Different
types
of
metal
efflux
systems
have
been
found
in
bacte-
http://dx.doi.org/10.1016/j.jbiotec.2015.09.032
0168-1656/©
2015
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/4.
0/).
172
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
ria.
Cupriavidus
metallidurans
CH34,
a
strain
able
to
growth
under
high
metal
concentrations,
contains
about
40
systems
involved
in
metal
detoxification,
including
P-type
ATPases
for
zinc
and
copper
extrusion,
RND
(resistance-nodulation-cell
division)
proteins
for
zinc,
copper,
cadmium,
cobalt,
nickel
and
silver
extrusion,
and
CDF
(cation
diffusion
facilitator)
systems
for
cobalt,
cadmium,
nickel
and
iron
extrusion
(Nies,
2003).
In
addition
to
the
physico-chemical
treatments
applied
to
remove
cyanide
from
industrial
residues,
biological
technologies
for
detoxification
of
free
cyanide
and
some
metal–cyanide
com-
plexes
have
been
described
(Akcil
and
Mudder,
2003;
Dash
et
al.,
2009).
Pseudomonas
pseudoalcaligenes
CECT5344
is
an
autochthonous
bacterium
that
was
isolated
from
the
Guadalquivir
River
(Córdoba,
Spain)
by
enrichment
cultivation
with
cyanide
(Luque-Almagro
et
al.,
2005a,b).
This
bacterial
strain
uses
free
(ion)
cyanide,
metal–cyanide
complexes
like
nitroferricyanide
(nitroprusside),
and
cyano-derivatives
like
cyanate
or
several
organic
cyanides
(nitriles)
as
the
sole
nitrogen
source
(Luque-Almagro
et
al.,
2005a,b;
Estepa
et
al.,
2012).
This
bacterium
is
able
to
grow
in
batch
reactor
with
sodium
cyanide
as
the
sole
nitrogen
source
under
alka-
line
conditions,
avoiding
cyanhydric
acid
volatilization
(Huertas
et
al.,
2010).
The
ability
of
the
strain
CECT5344
to
grow
with
the
metals-
and
cyanide-containing
jewelry
wastewater
was
also
demonstrated
(Luque-Almagro
et
al.,
2007).
All
these
characteris-
tics
make
this
bacterial
strain
a
suitable
candidate
to
be
applied
in
bioremediation
of
industrial
cyanide-containing
residues.
Cyanotrophic
microorganisms
display
different
cyanide
degra-
dation/detoxification
pathways,
including
hydrolytic,
oxidative
and
substitution/addition
reactions
(Gupta
et
al.,
2010;
Luque-
Almagro
et
al.,
2011a).
The
cyanide
degradation
pathway
of
P.
pseu-
doalcaligenes
CECT5344
includes
a
cytoplasmic
malate:quinone
oxidoreductase
that
converts
l-malate
into
oxaloacetate,
which
reacts
chemically
with
cyanide
to
produce
a
cyanohydrin
(nitrile)
that
is
transformed
into
its
corresponding
carboxylic
acid
and
ammonium
by
the
nitrilase
NitC
(Luque-Almagro
et
al.,
2011c;
Estepa
et
al.,
2012).
Recently,
the
whole
genome
sequence
of
P.
pseudoalcaligenes
CECT5344
has
been
elucidated
(Luque-Almagro
et
al.,
2013;
Wibberg
et
al.,
2014),
which
is
useful
to
perform
a
global
tran-
scriptomic
analysis.
Over
the
past
decades,
studies
on
cyanotrophic
microorganisms
have
been
focused
on
specific
genes
and/or
pro-
teins
required
for
cyanide
degradation.
This
study
describes
the
first
global
analysis
of
a
cyanide-assimilating
bacterial
strain,
P.
pseu-
doalcaligenes
CECT5344,
in
response
to
nitrogen
starvation,
sodium
cyanide
and
cyanide-
and
metal-containing
jewelry
wastewater
by
transcriptional
profiling
using
DNA
microarrays.
2.
Materials
and
methods
2.1.
Growth
of
P.
pseudoalcaligenes
CECT5344
P.
pseudoalcaligenes
cells
were
grown
in
minimal
medium
M9
(Sambrook
and
Russel,
2001)
containing
50
mM
sodium
acetate
as
carbon
source
and
2
mM
ammonium
chloride
as
nitrogen
source.
The
pH
of
the
media
was
adjusted
to
9.5.
The
strain
CECT5344
was
cultured
in
100
ml
flasks
filled
with
25
ml
of
media,
at
30 ◦C
and
continuous
agitation
at
220
rpm
on
a
shaker.
After
24
h,
when
ammonium
was
exhausted,
three
different
nitrogen
sources
were
added
to
different
cultures:
sodium
cyanide,
cyanide-containing
jewelry
wastewater
or
ammonium
chloride
(each
at
2
mM
concen-
tration).
10
ml-aliquots
were
harvested
by
centrifugation
at
4◦C
and
4000
×
g
for
5
min
when
cultures
reached
the
mid-exponential
growth
phase
and
the
remaining
nitrogen
source
in
the
media
was
about
30–50%
(Fig.
S1,
Supplementary
material).
An
additional
culture
was
set
up
with
a
low
amount
of
ammonium
chloride
(0.5
mM)
and
cells
were
harvested
under
the
above
described
conditions
15
min
after
ammonium
was
consumed
(nitrogen
star-
vation
condition).
Pellets
were
kept
at
−80 ◦C
until
use
and
four
independent
biological
replicates
for
each
nitrogen
source
were
carried
out.
The
Dps
mutant
strain
of
P.
pseudoalcaligenes
CECT5344
was
also
grown
in
100
ml
flasks
filled
with
25
ml
of
media
with
ammonium,
sodium
cyanide
or
jewelry
wastewater
(2
mM
concen-
tration).
When
indicated,
2
mM
FeCl3or
2
mM
CuCl2were
added
to
the
media.
The
wild-type
was
cultured
in
the
presence
of
nalidixic
acid
10
(g/ml)
and
the
Dps
mutant
strain
was
cultured
with
both
nalidixic
acid
10
(g/ml)
and
kanamycin
(25
g/ml).
2.2.
Total
RNA
isolation
The
frozen
aliquots
of
P.
pseudoalcaligenes
CECT5344
cell
mate-
rial
were
suspended
in
500
ml
lysozyme-containing
buffer
and
immediately
disrupted
using
the
RNAeasy
Midi
Kit
(Qiagen)
fol-
lowing
instructions
of
the
manufactures.
DNase
incubation
was
carried
out
in
the
column
with
RNase-free
DNase
set
(Qiagen)
and,
when
required,
an
additional
post-column
treatment
was
applied
with
DNase
I
(Ambion).
Quality
and
quantity
of
the
total
RNAs
were
checked
with
Bioanalyzer
(Agilent)
and
ND1000
spectrophotome-
ter
(Nanodrop
1000,
Agilent
Technologies-Wilmington,
DE,
USA).
All
samples
showed
an
integrity
number
(RIN)
higher
than
7.
2.3.
DNA
microarray
design
and
analysis
Oligonucleotides
(60
bp)
were
designed
for
4434
open
reading
frames
of
P.
pseudoalcaligenes
CECT5344
(Wibberg
et
al.,
2014)
with
the
software
eArray
(Agilent
Technologies).
Seven
probes
were
designed
for
each
gene.
RNA
quality
was
assessed
using
a
Tape
Station
(Agilent
Tech-
nologies).
RNA
concentration
and
dye
incorporation
was
measured
using
a
UV–vis
spectrophotometer
(Nanodrop
1000,
Agilent
Tech-
nologies).
Total
RNA
from
each
sample
was
reverse
transcribed,
labeled
and
hybridized
to
custom
microarray,
according
to
the
protocol
Two-Color
Microarray-Based
Prokaryote
Analysis
1.4,
Fair
Play
III
Labeling,
from
Agilent
Technologies.
Briefly,
reverse
tran-
scription
was
performed
with
AffinityScriptTM HC
RT
(Agilent
Technologies),
cDNA
was
labeled
with
a
chemical
coupling
method,
and
hybridized
following
protocol
of
the
manufacturers.
Dye
swaps
(Cy3
and
Cy5)
were
performed
from
each
sample.
Microarray
chips
were
washed
and
immediately
scanned
by
using
a
DNA
Microarray
Scanner
(Model
G2505C,
Agilent
Technologies).
Estimation
of
the
RNAm
from
different
samples
was
carried
out
previously
they
were
sent
to
Bioarrays,
S.L
(Alicante,
Spain)
for
microarrays
construction
and
consisted
of
q-PCR
analysis
of
all
RNAm
samples
by
using
the
three
housekeeping
genes,
the
␣-
subunit
of
the
DNA
polymerase
III
(BN5
2819),
the
-subunit
of
the
DNA
polymerase
III
(BN5
2215)
and
a
16S
rRNA
methyltransferase
(BN5
0873).
All
RNA
samples
showed
similar
expression
levels
for
all
these
genes.
Normalization
of
the
two
color
arrays
was
carried
out
by
intensity
normalization
with
the
MA
plot.
Gene
expression
analysis
was
carried
out
by
using
the
Fea-
ture
Extraction
Software
(v.
10.7)
available
from
Agilent,
using
the
default
variables.
Outlier
features
on
the
arrays
were
flagged
by
using
the
same
software
package.
Data
analysis
was
performed
with
a
Bioconductor
package,
under
R
environment.
Data
pre-
processing
and
differential
expression
analysis
was
performed
with
the
Limma
package
(htpp:/www.bioconductor.org/),
and
the
latest
gene
annotations
available
were
used.
Raw
feature
inten-
sities
were
background-corrected
with
Norme
xp
background
correction
algorithm.
Within-array
normalization
was
carried
out
by
using
Aquantile
normalization
and
spatial
and
intensity-
dependent
loess
method.
Gene
expression
was
reported
as
log2of
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
173
the
fold
change,
considering
≥2.1
fold
with
positive
values
(up-
regulated)
or
negative
values
(down-regulated)
and
statistically
significant
(p-value<0.01).
The
microarray
data
can
be
accessed
at
the
Gene
Expression
Omnibus
(GEO)
database
(accession
number
GSE69930).
The
functional
analysis
was
performed
by
using
GOStats
(Bio-
conductor),
which
identifies
significantly
enriched
GO
terms
among
a
list
of
genes
by
calculating
the
hypergeometric
probability
that
a
given
GO
term
is
represented
more
microarray
features
than
would
be
expected
by
chance.
Only
GOStats
results
with
a
p-value
<0.01
were
considered.
2.4.
Validation
of
DNA
microarray
data
P.
pseudoalcaligenes
CECT5344
cultures
and
RNA
isolations
were
performed
as
previously
described
for
DNA
arrays
generation.
The
concentration
and
purity
of
the
RNA
samples
were
mea-
sured
in
a
ND1000
spectrophotometer
(Nanodrop
1000,
Agilent
Technologies-Wilmington,
DE,
USA).
Synthesis
of
total
cDNA
was
achieved
in
20
ml
final
volume,
containing:
500
ng
RNA,
0.7
mM
dNTPs,
200
U
SuperScript
II
Reverse
Transcriptase
(Invitrogen)
and
3.75
mM
random
hexamers
(Applied
Biosystems).
Samples
were
initially
heated
at
65 ◦C
for
5
min
and
then
incubated
at
42 ◦C
for
50
min,
followed
by
incubation
at
70 ◦C
for
15
min.
To
carry
out
PCR
reactions,
2
ml
of
each
cDNA
were
initially
heated
at
98 ◦C
for
2
min,
followed
by
30
cycles
of
amplification:
98 ◦C,
30
s;
60 ◦C,
30
s
and
69 ◦C,
1
min.
Polymerase
extension
reactions
were
completed
by
an
additional
incubation
at
69 ◦C
for
10
min.
For
real-time
assays,
the
cDNA
was
purified
using
Favorprep
Gel/PCR
purification
kit
(Favorgen)
and
the
concentration
was
measured
using
a
ND1000
spectrophotometer.
The
iQ5
Multicolor
Real-Time
PCR
Detection
System
(Bio-Rad)
was
used
in
a
25
ml
reaction
(final
volume),
con-
taining
2
ml
diluted
cDNA
(12.5,
2.5
and
0.5
ng)
and
0.2
mM
of
each
primer
(Table
S1,
Supplementary
material),
and
12.5
ml
iQ
SYBR
Green
Supermix
(Bio-Rad).
Target
cDNAs
and
reference
sam-
ples
were
amplified
three
times
in
separate
PCR
reactions.
Samples
were
initially
denatured
by
heating
at
95 ◦C
for
3
min,
followed
by
40
cycles
of
amplification
(95 ◦C,
30
s;
test
annealing
temperature,
60 ◦C,
30
s;
elongation
and
signal
acquisition,
72 ◦C,
30
s).
For
rel-
ative
quantification
of
the
fluorescence
values,
a
calibration
curve
was
made
using
dilution
series
from
50
to
0.0005
ng
of
P.
pseu-
doalcaligenes
CECT5344
genomic
DNA
sample.
Represented
data
were
normalized
by
using
16S
rRNA
methyltransferase
(BN5
0873),
23S
rRNA
methyltranferase
(BN5
2541),
DNA
polymerase
III
␣-
subunit
(BN5
2819)
and
DNA
polymerase
III
-subunit
(BN5
2215)
as
housekeeping
genes
(Table
S1,
Supplementary
material).
Error
bars
represent
standard
deviation
calculated
from
the
results
of
three
independent
experiments.
2.5.
Generation
of
a
Dps
mutant
of
P.
pseudoalcaligenes
CECT5344
A
mutant
strain
of
P.
pseudoalcaligenes
CECT5344
was
generated
by
insertion
of
a
kanamycin
cassette
in
the
dps
gene
(BN5
3091)
that
codes
for
a
ferritin-like
protein
in
this
bacterium.
Genomic
DNA
was
isolated
as
indicated
by
the
protocol
of
manufacturers
(Wizard
Genomic
Purification
Kit,
Promega).
The
dps
gene
was
amplified
from
genomic
DNA
by
PCR
using
the
oligonucleotides:
Dps
F:
5-
AGCGGATCCTCTGTACCTGAAGACCCATAACTTC-3
(BamHI
site
is
underlined)
and
Dps
R:
5-
AGCAAGCTTCGTTTGTTAAGTAAACGGCATTAC-3(HindIII
site
is
underlined)
to
yield
a
450
bp
DNA
fragment.
The
PCR
program
consisted
of
an
initial
denaturing
step
by
heating
at
95 ◦C
for
3
min,
followed
by
40
cycles
of
amplification
(95 ◦C,
30
s;
test
annealing
temperature,
65 ◦C,
30
s;
elongation
and
signal
acquisition,
72 ◦C,
30
s).
The
PCR
fragment
was
cloned
into
pBluescrip-SK
vector
with
the
restriction
sites
BamHI
and
HindIII.
The
kanamycin
(Km)
cassette
was
inserted
to
disrupt
the
dps
gene
by
using
a
SphI
restriction
site
located
at
200
bp
from
the
5-end
of
the
dps
gene.
The
dps::Km
fragment
was
cloned
into
the
mobilizable
vector
pK
18mob
previously
digested
with
the
restriction
enzymes
BamHI
and
HindIII.
This
mobilizable
construct
was
transferred
to
the
wild-type
strain
by
conjugation
and
transconjugants
were
selected
by
homologous
recombination
in
media
with
nalidixic
acid
and
kanamycin.
3.
Results
The
alkaliphilic
bacterium
P.
pseudoalcaligenes
CECT5344
uses
free
cyanide
(CN−)
and
different
metal–cyanide
complexes
as
the
sole
nitrogen
source
for
growth
(Luque-Almagro
et
al.,
2005a,b),
and
the
whole
genome
of
this
bacterium
has
been
sequenced
and
annotated
(Luque-Almagro
et
al.,
2013
Wibberg
et
al.,
2014).
To
obtain
insights
into
the
effect
of
different
nitrogen
sources,
includ-
ing
cyanide
and
a
cyanide-rich
industrial
residue,
DNA
microarrays
from
P.
pseudoalcaligenes
CECT5344
cells
grown
with
different
nitrogen
sources
have
been
constructed.
DNA
microarrays
from
cells
grown
with
2
mM
sodium
cyanide
(NaCN)
or
cyanurated
wastewater
(CN-WW)
from
the
jewelry
industry
(2
mM
cyanide
concentration)
were
compared
against
microarrays
from
cultures
with
2
mM
ammonium
(NH4Cl)
as
nitrogen
source.
A
condition
of
nitrogen
starvation
(−N)
was
also
compared
against
DNA
microar-
rays
from
cultures
with
ammonium.
Four
independent
biological
replicates
for
each
nitrogen
source/condition
have
been
used
to
carry
out
the
microarrays
con-
struction
by
Agilent
Technologies,
as
previously
and
successfully
used
in
other
bacterial
transcriptomic
studies
(Esclapez
et
al.,
2015).
The
linkage
of
the
four
independent
biological
replicates
for
each
nitrogen
source/condition
was
statistically
analyzed
by
different
methods,
including
the
Ward’s
linkage
method
(Fig.
S2,
Supple-
mentary
material).
As
expected,
samples
from
the
same
nitrogen
source
were
closer
within
them
than
to
the
samples
from
other
conditions.
In
addition,
cultures
with
cyanide
(sodium
cyanide
or
cyanide-containing
industrial
wastewater)
were
closer
between
them
than
to
those
from
different
nitrogen
sources.
Samples
from
ammonium,
the
preferential
nitrogen
source
for
many
organisms,
were
more
distant
to
samples
from
cyanide-containing
cultures.
The
whole
genome
of
the
strain
CECT5344
includes
4434
pro-
tein
coding
sequences
(Wibberg
et
al.,
2014).
Among
them,
a
total
of
1783
genes
(40%)
showed
expression
changes
in
the
culture
con-
ditions
used
in
this
study,
with
862
up-regulated
genes
and
921
down-regulated
genes
(Fig.
1).
Genes
were
considered
differen-
tially
expressed
when
they
fulfilled
the
following
filter
parameters:
expression
ratio
log2fold
change
≥2.1
with
positive
value
for
up-
regulated
genes
or
negative
value
for
down-regulated
genes,
and
an
adjusted
p-value≤0.01.
3.1.
P.
pseudoalcaligenes
CECT5344
response
to
nitrogen
starvation
conditions
The
limitation
of
nitrogen
leaded
considerable
changes
in
the
DNA
arrays
of
P.
pseudoalcaligenes
CECT5344
when
com-
pared
against
microarrays
from
ammonium
grown-cells.
A
total
of
692
genes
(15.6%)
were
found
up-regulated
in
nitrogen-starved
cells
whereas
753
genes
(17%)
were
down-regulated.
Among
up-
regulated
genes,
399
genes
were
specifically
induced
by
nitrogen
limitation
and
293
genes
were
also
induced
by
cyanide
and/or
the
jewelry
residue.
Likewise,
352
genes
were
specifically
down-
regulated
in
nitrogen
starved
cells
and
401
genes
were
also
repressed
by
the
presence
of
cyanide
(Fig.
1).
Induced
genes
by
nitrogen
starvation
can
be
grouped
in
different
functional
(over-
174
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
Table
1
P.
pseudoalcaligenes
CECT5344
most
relevant
genes
specifically
affected
by
nitrogen
starvation
compared
against
ammonium.
Gene
IDaAnnotation
(function/gene
name) log2FC p-Value
BN5
0139
ABC-type
amino
acid
transport/signal
transduction
systems,
periplasmic
component
(glnH)
2.225
2.0E
−
11
BN5
0140
ABC-type
amino
acid
transport
system,
permease
component
(glnP1)
2.564
2.1E
−
11
BN5
0141
ABC-type
amino
acid
transport
system,
permease
component
(glnP3)
1.379
2.0E
−
09
BN5
1009
ABC-type
polar
amino
acid
transport
system,
ATPase
component
(aapP)
1.162
4.2E
−
08
BN5
0142
ABC-type
polar
amino
acid
transport
system,
ATPase
component
(glnQ)
2.101
9.1E
−
10
BN5
3231
ABC-type
branched-chain
amino
acid
transport
systems,
periplasmic
component
2.649
2.5E
−
11
BN5 3113 Cyanate
permease
(cynX) 1.117 1.1E
−
06
BN5
0499
Membrane
transporter
of
cations
and
cationic
drugs
1.582
9.5E
−
08
BN5
2691
Permease
of
the
drug/metabolite
transporter
(DMT)
superfamily
1.277
3.0E
−
07
BN5
2416
Fe2+-dicitrate
sensor,
membrane
component
(fecR)
1.244
5.7E
−
05
BN5
3557
ABC-type
multidrug
transport
system,
ATPase
component
1.333
3.2E
−
08
BN5
3747
Mn2+ and
Fe2+ transporter
of
the
NRAMP
family
1.292
2.5E
−
06
BN5 4476 Mercuric
ion
transport
(merT5) 1.983 2.1E
−
10
BN5 1137 Ammonia
permease
(amt) 1.984 3.4E
−
10
BN5
0438
Transcriptional
regulator
containing
PAS
and
DNA-binding
domain
(cynF)
1.419
7.2E
−
07
BN5
0178
Nitrogen
regulatory
protein
PII
(glnK)
3.488
8.6E
−
13
BN5
3961
NtrB
signal
transducer,
responds
to
the
nitrogen
level
and
modulates
NtrC
activity
(ntrB)
2.728
7.8E
−
11
BN5 3962 Nitrogen
response
regulator
containing
DNA-binding
domain
(ntrC3)
2.596
4.9E
−
13
BN5
4425
Transcriptional
regulator.
Nodulation
protein
D1
(mexT5)
1.332
1.6E
−
06
BN5
0781
Transcriptional
regulators
(MocR
family)
1.621
5.2E
−
09
BN5
2516
Glutamate
synthase
1.713
1.5E
−
09
BN5
2542
Aconitase
A
(acnA)
1.081
2.6E
−
06
BN5 1500 Carbon
storage
regulator,
Global
regulator
protein
family
(csrA) 1.418 9.3E
−
06
BN5
3204
Aliphatic
amidase
with
a
restricted
substrate
specificity,
hydrolyzes
formamide
(amiF)
2.373
8.3E
−
13
BN5
3668
Bacterioferritin
(bfr)
1.507
1.2E
−
09
BN5
0360
Glutathione
S-transferase
1.464
1.5E
−
07
BN5
1595
Glutathione
peroxidase
1.319
2.5E
−
08
BN5
0763
CRISPR-associated
helicase
Cas3
involved
in
defense
mechanisms
1.251
3.7E
−
08
BN5
0150
Cytochrome
c,
mono-
and
diheme
variants
(cccA)
1.115
4.2E
−
08
BN5 1586 Cobalamin
biosynthesis
protein
(cobD/cbiB)
1.591
2.3E
−
08
BN5
4374
Cobalamin
biosynthesis
protein
(cobK)
1.218
4.3E
−
08
BN5
3669
Catalase-peroxidase
I
(katG)
1.563
4.0E
−
10
BN5
1467
Molybdenum
cofactor
biosynthesis
enzyme
(moaA1)
1.177
3.3E
−
09
BN5
2778
Uncharacterized
protein
involved
in
formation
of
periplasmic
nitrate
reductase
(napD)
1.258
9.1E
−
06
BN5 0412 Poly(3-hydroxyalkanoate)
synthetase
(phaC2) 2.358 5.6E
−
10
BN5
0410
Poly(hydroxyalcanoate)
granule
associated
protein
(phaF1)
3.355
7.1E
−
13
BN5
4096
Poly(hydroxyalcanoate)
granule
associated
protein
(phaP)
1.874
2.3E
−
09
BN5
0413
Predicted
hydrolase
or
acyltransferase
(alpha/beta
hydrolase
superfamily)
(phaZ)
1.554
4.3E
−
09
BN5
3102
SAM-dependent
methyltransferase
(ubiG3)
1.465
6.5E
−
09
BN5
0581
Urea
amidohydrolase
(urease)
gamma
subunit
(ureA)
1.814
1.9E
−
10
BN5
0578
Urea
amidohydrolase
(urease)
alpha
subunit
(ureC)
1.443
9.5E
−
10
BN5 0554 Putative
gene
involved
in
urea
metabolism
(ureE) 2.946 6.9E
−
13
BN5
0551
Hydrogenase/urease
accessory
gene
(ureJ)
2.328
2.1E
−
11
BN5
4078
ABC-type
polar
amino
acid
transport
system,
ATPase
component
−2.215
7.8E
−
13
BN5
4080
ABC-type
amino
acid
transport
system,
permease
component
−1.646
2.5E
−
07
BN5
4081
ABC-type
amino
acid
transport
system,
permease
component
−1.667
5.8E
−
07
BN5
0814
Cation/multidrug
efflux
pump
(ttgB)
−1.298
2.5E
−
06
BN5
2733
Predicted
exporter
of
the
RND
superfamily
−1.858
6.8E
−
08
BN5
2616
ABC-type
transport
system
involved
in
cytochrome
c
biogenesis
(ccmC)
−1.078
7.1E
−
07
BN5
4325
ABC-type
metal
ion
transport
system,
permease
component
(metI)
−1.189
2.8E
−
05
BN5
4326
ABC-type
metal
ion
transport
system,
periplasmic
component
(metQ)
−3.823
1.8E
−
10
BN5
0341
Predicted
transcriptional
regulator
(arsR3)
−1.396
4.6E
−
06
BN5
2792
Phosphoribosylaminoimidazole
(AIR)
synthetase
(purM)
−1.544
1.8E
−
05
BN5
1068
Formate-dependent
phosphoribosylglycinamide
formyltransferase
GAR
(purT)
−1.420
9.5E
−
09
BN5
4231
NAD(P)H-nitrite
reductase
(rubB)
−2.014
1.7E
−
09
BN5
1676
Phosphoserine
aminotransferase
(serC1)
−1.538
2.5E
−
10
BN5
2026
Sulfite
reductase,
beta
subunit
(sir)
−2.507
6.2E
−
12
BN5
3036
Peroxiredoxin
(ahpC1)
−1.664
1.3E
−
08
BN5
3035
Alkyl
hydroperoxide
reductase,
large
subunit
(ahpF)
−1.085
2.8E
−
07
BN5
2615
Putative
gene
involved
in
cytochrome
c
biogenesis
(ccmD)
−1.256
2.4E
−
07
BN5
2614
Cytochrome
c-type
biogenesis
protein
(ccmE)
−1.557
6.7E
−
11
BN5
2613
Cytochrome
c
biogenesis
factor
(ccmF)
−1.643
3.3E
−
11
BN5
2610
Cytochrome
c
biogenesis
factor
(ccmH)
−2.109
2.9E
−
11
BN5 1342 Cbb3-type
cytochrome
oxidase,
subunit
1
(ccoN1)−1.259
9.6E
−
07
BN5
4400
Precorrin-4
methylase
(cobM)
−1.153
2.8E
−
04
BN5
1592
Cobalamin-5-phosphate
synthase
(cobS)
−1.109
7.9
E
−
05
BN5
0743
Putative
Mg2+ and
Co2+ transporter
(corC)
−1.183
5.2
E
−
08
BN5
0325
5,10-methylenetetrahydrofolate
reductase
(metF1)
−2.022
2.5E
−
10
BN5
2035
Methionine
synthase
I,
cobalamin-binding
domain
(metH)
−1.207
5.1E
−
07
aGene
IDs
refer
to
accession
number
HG916826
(Wibberg
et
al.,
2014).
represented
GO)
categories
according
to
GOStats
analysis
(Figs.
S3
and
S4,
Supplementary
material),
including
genes
that
code
for
DNA
polymerase
activity,
motor
activity,
nucleic
acid
binding,
nitrogen
cycle
and
regulation
of
metabolic
processes.
The
up-
regulated
genes
exclusively
induced
in
nitrogen-starved
cells
were
related
to
transport
of
amino
acids,
cyanate
or
ammonia,
amino
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
175
Fig.
1.
Venn
diagram
with
the
number
of
up-
and
down-regulated
genes
in
the
transcriptomes
of
P.
pseudoalcaligenes
CECT5344
from
sodium
cyanide
(NaCN),
cyanide-containing
jewelry
wastewater
(CN-WW)
and
nitrogen-limiting
conditions
compared
against
ammonium.
An
expression
ratio
of
log2fold
≥2.1
with
positive
values
(up-regulated
genes)
and
negative
values
(down-regulated
genes)
and
an
adjusted
p-value
of
≤0.01
were
considered.
acid
metabolism,
regulation
of
nitrogen
metabolism
processes
like
global
nitrogen
control
or
cyanate
and
urea
metabolism,
enzymes
and
proteins
involved
in
polyhydroxyalkanoate
metabolism,
and
genes
related
to
defense
mechanisms,
iron
acquisition,
metal
transport
and
oxidative
stress,
such
as
glutathione
peroxidase,
CRISPR-associated
helicase,
catalase,
bacterioferritin
and
the
FecR
Fe2+-dicitrate
sensor
(Table
1).
Many
genes
were
found
down-regulated
in
the
nitrogen
star-
vation
response.
Most
of
the
specifically
down-regulated
genes
were
involved
in
amino
acids
and
metal
transport,
methionine
metabolism,
cytochrome
c
biogenesis
and
sulfite/nitrite
reduc-
tion
(Table
1).
Expression
of
genes
encoding
ribosomal
proteins,
tRNA
aminoacylation
proteins
for
protein
translation
and
transla-
tion
factors
was
also
decreased
(Tables
S2,
S6–S8,
Supplementary
material).
3.2.
P.
pseudoalcaligenes
CECT5344
response
to
sodium
cyanide
and
a
cyanide-containing
industrial
wastewater
The
presence
of
cyanide
provoked
relevant
changes
in
the
tran-
scripts
of
the
strain
CECT5344.
Two
different
sources
of
cyanide
were
considered
as
nitrogen
source
for
growth,
and
consequently,
two
different
DNA
microarrays
were
obtained
and
compared
against
microarrays
from
ammonium
cultures:
sodium
cyanide
(NaCN)
that
dissociates
in
aqueous
solution
to
free
ion
cyanide,
and
cyanide-containing
wastewater
from
the
jewelry
industry
(CN-
WW),
which
contains
both
free
ion
cyanide
and
stable
cyano–metal
complexes.
Only
37
or
34
genes
(0.8%
genes
in
the
whole
genome)
were
specifically
induced
or
repressed,
respectively,
in
response
to
sodium
cyanide
(Fig.
1).
Some
genes
exclusively
up-regulated
in
sodium
cyanide
code
for
acyl-CoA
transferases,
aminotrans-
ferases
and
deamidases,
a
heme/copper-type
cytochrome/quinol
oxidase
subunit,
and
the
Nit3
nitrilase/cyanide
hydratase
(Table
S3,
Supplementary
material).
These
cyanide-induced
genes
can
be
grouped
in
different
functional
categories
by
GOStats
analysis
(Figs.
S5
and
S6,
Supplementary
material)
and
include
genes
involved
in
metal-
and
iron–sulphur
cluster
binding,
pyrydoxal
phos-
phate
cofactor
binding,
enzymes
displaying
oxidoreductase
activity
(l-serine
family
metabolic
process)
and
hydroxymethyl-formyl
and
related
transferases.
Among
genes
specifically
repressed
by
cyanide
were
highlighted
those
related
to
Fe-S
oxidoreductases,
metal-dependent
hydrolases
and
cytochrome
c
biogenesis
(Table
S3,
Supplementary
material).
The
GOStats
functional
categories
according
to
GOStats
analysis
for
down-regulated
genes
in
sodium
cyanide
are
related
to
ATPase
activity,
C–C
and
C–N
ligase
activity,
transport
of
ions
and
RNA
binding.
Transcription
of
128
genes
(2.9%
of
total
genes
in
the
whole
genome)
was
specifically
affected
in
response
to
the
cyanide-
containing
jewelry
wastewater,
with
57
(1.3%)
induced
and
71
(1.6%)
repressed
genes
(Fig.
1).
The
up-regulated
genes
in
the
jew-
elry
residue
are
shown
in
Table
S4
(Supplementary
material),
and
including
genes
for
metal
transporters,
such
as
Fe3+-siderophore
ABC-type
transporter,
P-type
ATPases
for
Cu+or
Cd2+ extrusion
and
multidrug
efflux
pumps
from
the
RND
family,
regulatory
genes
involved
in
metal
detoxification
systems,
and
genes
encod-
ing
arsenate
reductase,
d-methionine
ABC-type
transporter
and
malate:quinone
oxidoreductase
B
(mqoB).
According
to
the
GOStats
analysis,
genes
induced
by
the
jewelry
residue
can
be
grouped
in
functional
categories
similar
to
those
described
for
genes
affected
by
sodium
cyanide
(Figs.
S7
and
S8,
Supplementary
material),
which
were
relates
to
iron–sulphur
and
copper
cluster
binding,
pyrydoxal
phosphate
binding,
inorganic
anion
transport,
and
oxi-
doreductases
(l-serine
family
metabolic
process-related
enzymes).
The
GOStats
functional
categories
for
the
down-regulated
genes
by
the
jewelry
residue
were
related
to
ATPase
activity,
racemase
and
ligase
activities,
acyl-transferase
activities,
transport
of
ions
and
RNA
and
NAD
binding.
Some
genes
specifically
repressed
by
the
jewelry
liquid
residue
were
the
carbon–nitrogen
hydrolase
(nitri-
lase
family)
aguB
gene,
the
DNA-binding
ferritin-like
dps
gene,
the
iron
and
zinc
uptake
regulator
(zur)
gene,
and
genes
encod-
ing
citrate
synthase,
acyl-CoA
dehydrogenases
and
uncharacterized
flavoenzymes
(Table
S4,
Supplementary
material).
A
Dps
mutant
strain
of
P.
pseudoalcaligenes
CECT5344
has
been
generated
by
insertion
of
a
kanamycin
antibiotic
resistance
cassette.
Growth
of
Dps
mutant
strain
in
the
presence
of
a
high
concentration
of
CuCl2
(2
mM),
with
ammonium
as
nitrogen
source,
was
much
lower
than
growth
of
the
wild-type
strain.
However,
wild-type
and
Dps
mutant
strains
showed
similar
growth
in
the
presence
of
2
mM
FeCl3and
ammonium.
In
cyanide-containing
media,
the
Dps
mutant
of
P.
pseudoalcaligenes
CECT5344
strain
also
showed
a
similar
growth
to
the
wild-type
strain
(not
shown).
Expression
of
139
genes
was
affected
in
cells
grown
with
sodium
cyanide
or
with
cyanide-containing
jewelry
wastewater
when
compared
against
ammonium
(Table
S5).
Induced
genes
(a
total
of
76)
were,
among
others,
two
nitrilase-encoding
genes
(nit2
and
nit4),
sulfur
metabolism
genes
like
those
coding
for
sulfite
reductase
and
for
sulfate
and
sulfonates
transporters,
the
cioB
gene
coding
for
a
terminal
cbb3-type
oxidase
and
the
ccoN1
and
cyoA
oxidase
genes,
several
amino
acid
metabolism
genes
that
are
clustered
together
the
cioAB
genes
and
encode
putative
176
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
aminotransferases
for
serine,
histidine
and
arginine,
genes
encod-
ing
methionine
synthases
vitamin
B12-dependent
or
-independent,
the
alkyl-hydroperoxide
reductase
ahpC
gene
that
is
adjacent
to
the
sulfur
metabolism
genes,
the
Hmp
flavohaemglobin
fpr
gene,
the
isc
genes
for
Fe-S
cluster
assembly
system,
and
the
malic
enzyme
gene.
The
down-regulated
genes
presented
differ-
ent
functions,
and
included
those
coding
for
RNA
polymerase
and
CRISPR/Cas-associated
proteins
for
defense
mechanisms
(Table
S5,
Supplementary
material).
A
total
of
77
genes
(1.7%)
were
found
affected
in
common
in
the
P.
pseudoalcaligenes
CECT5344
DNA
microarrays
from
cells
grown
with
sodium
cyanide
or
under
nitrogen-limiting
condi-
tions
when
both
compared
against
ammonium.
Transcripts
with
27
genes
(0.6%)
were
up-regulated
whereas
50
genes
(1.1%)
were
down-regulated
(Fig.
1).
The
up-regulated
genes
(Table
S6,
Supple-
mentary
material)
included,
among
others,
the
heme/copper-type
cytochrome/quinol
oxidase
(cox3)
and
the
aconitase
A
(acnC)
genes.
Several
down-regulated
genes
code
for
succinate
dehydro-
genase
subunits,
efflux-type
ATPase
and
RND
efflux
pump
(mexC),
glutamine
synthetase
(glnA1)
and
a
rhodanase-related
sulfurtrans-
ferase.
The
DNA
microarrays
of
P.
pseudoalcaligenes
CECT5344
in
response
to
the
jewelry
wastewater
and
nitrogen-limiting
condi-
tions,
both
compared
against
ammonium,
shared
a
total
of
195
affected
genes
(4.4%),
with
100
induced
(2.3%)
and
95
(2.1%)
repressed
genes
(Fig.
1;
Table
S7,
Supplementary
material).
Rele-
vant
induced
genes
were
involved
in
nitrate/nitrite
assimilation,
including
the
two-component
positive
regulatory
system
that
responds
to
nitrate
and/or
nitrite
(nasT
and
nasS
genes),
the
nitrate
and
nitrite
transporters,
and
the
catalytic
nitrate
and
nitrite
reductases,
some
regulatory
genes
like
the
metal
regulator
gene
cueR
and
the
polyhydrohyalkanoate
structural
and
regulatory
gene
phaI,
metal
transporters
like
a
Ni-transporter
and
a
Cu-
trans-
porting
ATPase
(actP
gene)
and
a
Zn–Co–Cd
efflux
pump,
and
ABC-type
transporters
for
amino
acids,
sugar
and
other
compounds.
Down-regulated
genes
code
for
aconitase
B
acnB
gene,
acetyl-CoA
dehydrogenases,
aminotransferases,
biotin
cofactor
biosynthesis,
sulfate
transporters
and
glutamate
synthase
(gltB1).
DNA
microarrays
of
P.
pseudoalcaligenes
CECT5344
in
response
to
sodium
cyanide,
jewelry
wastewater
and
nitrogen-limiting
con-
ditions,
all
three
compared
against
ammonium,
shared
a
total
of
422
affected
genes
(9.5%),
with
166
up-regulated
(3.7%)
and
256
(5.8%)
down-regulated
genes
(Fig.
1;
Table
S8,
Supplementary
material).
Some
repressed
genes
code
for
aspartate
kinase,
ATPase
subunits,
cytochrome
c
oxidases,
citrate
synthase
and
oxaloac-
etate
decarboxylase
(Table
2).
Relevant
genes
induced
in
all
three
experimental
conditions
tested
were
the
nitrilase
nitC
gene
that
is
essential
for
cyanide
assimilation
in
the
strain
CECT5344
and
other
genes
of
unknown
function
that
constitute
the
nit1C
transcrip-
tional
unit,
genes
encoding
GntR–MocR
and
pyridoxal
phosphate
(PLP)-dependent
aminotransferases,
biotin
transport
and
synthesis
genes,
several
genes
that
are
usually
up-regulated
under
nitrogen-
limiting
conditions
like
those
coding
for
glutamine
synthetase,
glutamate
synthase
and
ammonium
transporter,
genes
for
the
ISC
system
for
Fe-S
cluster
assembly,
and
genes
coding
for
the
ABC-
type
cyanate
transporter
(cynABD)
and
the
cyanase
(cynS)
involved
in
cyanate
assimilation.
3.3.
qRT-PCR
validation
of
microarray
data
Microarray
data
were
corroborated
by
quantitative
RT-PCR
gene
expression
of
P.
pseudoalcaligenes
CECT53344
selected
genes,
like
the
nitrilase-encoding
genes
(nit2/nitC,
nit3,
nit4
and
aguB)
and
the
cioA
and
cioB
genes
coding
for
the
cyanide-insensitive
termi-
nal
oxidase
(Fig.
2).
The
genome
of
P.
pseudoalcaligenes
CECT5344
contains
four
nitrilase
genes,
nit1
(BN5
0736),
nit2/nitC
(BN5
1632),
nit3
(BN5
3251)
and
nit4
(BN5
1912),
although
only
the
nitC
gene
is
essential
for
cyanide
assimilation
(Estepa
et
al.,
2012).
These
four
nitrilase-encoding
genes
have
been
found
to
be
differen-
tially
expressed
in
the
nitrogen
conditions
used
in
this
study.
In
addition
to
these
four
nitrilase
genes,
an
additional
gene,
aguB,
which
belongs
to
the
nitrilase/C–N
hydrolase
superfamily
was
also
affected
in
the
DNA
microarrays.
Under
nitrogen
liming
condi-
tions,
expression
of
all
nitrilase
genes
was
very
low,
except
the
nitC
gene
that
was
expressed
at
a
high
level.
In
cells
grown
with
cyanide-containing
media
(sodium
cyanide
or
jewelry
residue),
all
nitrilase
genes
increased
their
expression,
except
aguB
gene
that
was
repressed.
Repression
of
aguB
gene
was
higher
in
cells
grown
with
the
jewelry
residue
than
with
sodium
cyanide.
These
results
confirmed
the
microarray
data
since
the
nit3
gene
was
induced
exclusively
by
sodium
cyanide,
the
nit4
gene
was
induced
by
both
sodium
cyanide
and
cyanide-containing
jewelry
wastewater,
the
nitC
gene
was
induced
by
sodium
cyanide
and
cyanide-containing
wastewater
and
also
under
nitrogen
starvation,
and
the
aguB
gene
was
repressed
by
the
jewelry
residue.
Likewise,
the
microarrays
analysis
revealed
that
the
cioAB
genes
were
induced
in
the
pres-
ence
of
cyanide
(sodium
cyanide
and
jewelry
residue),
displaying
concordance
with
their
expression
profiles
found
by
qRT-PCR
anal-
ysis.
4.
Discussion
DNA
microarrays
have
been
constructed
from
cells
grown
with
sodium
cyanide
or
cyanide-containing
wastewater
from
the
jewelry
industry
and
compared
against
DNA
microarrays
from
cul-
tures
with
ammonium
chloride
as
nitrogen
source.
Additionally,
microarrays
from
cultures
under
nitrogen
starvation
have
been
also
carried
out.
Microarrays
analysis
has
been
previously
applied
to
study
the
response
to
nitrogen
starvation
in
several
bacterial
strains
like
the
Gram
positive
Corynebacterium
glutamicum
(Silberbach
et
al.,
2005),
the
Gram
negative
Pseudomonas
putida
(Hervás
et
al.,
2008),
and
the
haloarchaeon
Haloferax
mediterranei
(Esclapez
et
al.,
2015).
Although
the
response
to
a
very
low
concentration
of
cyanide
(1
mM)
has
been
studied
in
Nitrosomonas
europaea,
this
strain
lacks
of
a
cyanide
assimilatory/detoxification
pathway
(Park
and
Ely,
2009).
Therefore,
there
are
no
data
available
about
transcriptomic
studies
of
the
response
to
cyanide
in
a
cyanotrophic
microorganism.
DNA
microarrays
revealed
that
large
number
of
genes
were
up-
regulated
in
cells
grown
under
nitrogen
starvation
when
compared
against
DNA
microarrays
from
ammonium
grown
cells
(Fig.
1).
It
has
been
previously
described
that
under
nitrogen-limiting
condi-
tions
P.
putida
induces
NtrC-dependent
genes,
including
amino
acid
transport
and
urea
assimilation
genes,
the
PII-encoding
glnK
gene
and
the
ammonium
transporter
amtB
gene
(Hervás
et
al.,
2008).
Under
nitrogen
deprivation,
carbon
storage
is
potentiated
as
sug-
gested
by
the
induction
of
polyhydroxyalkanoates
synthase
and
phasin
genes
(Hervás
et
al.,
2008).
These
genes
were
also
induced
in
P.
pseudoalcaligenes
CECT5344
cells
subjected
to
nitrogen
starva-
tion
(Table
1).
Interestingly,
the
carbon
storage
regulator
csrA
gene
was
also
induced
by
nitrogen
starvation
in
the
strain
CECT5344.
In
Escherichia
coli,
it
has
been
described
that
the
csrA
gene
acts
as
a
repressor
by
mRNA
binding,
avoiding
translation
(Gutiérrez
et
al.,
2005).
This
could
be
a
mechanism
to
prevent
an
excess
of
catabolic
flow
through
TCA
cycle
under
nitrogen
limiting
condi-
tion
for
growth.
GOStats
analysis
of
P.
pseudoalcaligenes
CECT5344
microarrays
revealed
a
reduced
expression
of
genes
encoding
some
ribosomal
proteins,
translation
factors
and
tRNA
aminoacylation
proteins
(Tables
S2,
S6–S8,
Supplementary
material),
indicating
that
there
is
a
general
decrease
of
protein
synthesis
under
nitrogen
limiting
conditions.
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
177
Table
2
P.
pseudoalcaligenes
CECT5344
most
relevant
genes
affected
by
sodium
cyanide,
jewelry
wastewater
and
nitrogen
starvation
compared
against
ammonium.
Gene
IDaAnnotation
(function/gene
name)
CN−-NH4+log2FC
WW-NH4+log2FC
N-NH4+log2FC
CN-NH4+p-value
WW-NH4+p-value
N-NH4+p-value
BN5
1902
Cytochrome
bd
ubiquinol
oxidase
(cioA3)
8.128
7.702
1.151
3.1E
−
12
3.0E
−
12
2.9E
−
3
BN5 1634 GCN5-related
N-acetyltransferase 7.653 9.954
7.164
1.8E
−
12
1.2E
−
13
4.5E
−
12
BN5
1632
NitC,
Nitrilase/cyanide
hidratase
(nit2/nitC)
6.482
9.394
6.292
9.9E
−
11
8.0E
−
13
1.3E
−
10
BN5
0442
Cyanate
lyase,
cyanate
hydratase
(cynS)
6.243
9.156
4.112
2.7E
−
07
1.1E
−
09
8.4E
−
06
BN5
1899
PLP-dependent,
GntR-regulator
(mocR)
6.041
5.912
1.574
4.5E
−
13
3.2E
−
13
1.6E
−
06
BN5
1353
Cytochrome
c
oxidase
cbb3
type
(ccoG)
5.804
5.879
1.618
2.2E
−
14
1.4E
−
14
1.0E
−
08
BN5
0439
ABC-type
cyanate
transporter
(cynA)
5.695
10.330
5.867
9.6E
−
08
2.9E
−
11
4.2E
−
08
BN5
1892
LysR,
substrate-binding
5.311
5.226
1.520
1.0E
−
12
6.8E
−
13
9.4E
−
07
BN5 0441 ABC-type
cyanate
transporter
(cynD) 5.152 9.356 5.351 2.6E
−
08
8.3E
−
12
6.6E
−
09
BN5
2689
Pyruvate
ferredoxin/flavodoxin
(iorA1)
4.580
4.226
1.666
2.8E
−
10
4.3E
−
10
1.4E
−
05
BN5
2413
PLP-dependent
aminotransferase
4.553
4.546
4.007
1.3E
−
07
1.3E
−
08
1.7E
−
07
BN5
0440
ABC-type
cyanate
transporter
(cynB)
4.202
8.812
4.185
3.1E
−
06
1.9E
−
10
9.2E
−
07
BN5
0329
Glutamate
synthase-GOGAT
(gltD1)
2.907
3.087
5.773
2.7
E
−
4
1.5
E
−
4
8.5E
−
08
BN5
1638
Isocitrate
dehydrogenase
(aceK)
2.523
3.782
1.789
2.7E
−
06
1.2E
−
08
7.8E
−
06
BN5
2309
Glutamine
synthetase-GS
(glnA5)
2.383
2.512
3.199
1.6E
−
07
5.3E
−
08
1.6E
−
10
BN5
2268
Biotine
synthase
2.195
1.037
1.063
1.0E
−
08
2.5E
−
05
1.3E
−
05
BN5
2535
Bacterial
regulatory
protein,
MarR
2.160
2.427
3.093
7.6E
−
10
9.4E
−
11
1.1E
−
11
BN5 2414 Bacterial
regulatory
protein,
MarR 2.068 2.456 1.661
5.2E
−
08
3.3E
−
09
1.6E
−
07
BN5
1587
PLP-dependent
aminotransferase
(cobC)
2.068
3.852
1.357
5.6E
−
08
1.2E
−
11
2.1E
−
06
BN5
1338
LysR,
GntR
transcriptional
regulators
(gstR)
1.868
1.620
2.122
9.5E
−
07
2.3E
−
06
6.7E
−
08
BN5
3729
FeS
cluster
assembly
1.762
2.440
1.579
1.3E
−
06
7.6E
−
09
7.2E
−
07
BN5
3269
ISC
system
trasnscription
regulator
(iscR)
1.714
2.718
1.667
7.5E
−
06
2.0E
−
08
3.1E
−
06
BN5
1807
Biotin/lipoyl,
RND
family
efflux
transporter
1.494
1.003
1.244
2.2E
−
4
2.2E
−
05
1.5E
−
06
BN5
2308
PLP-dependent
aminotransferase
1.410
1.705
1.488
3.5E
−
07
1.9E
−
08
5.9E
−
08
BN5 0594 Bacterial
regulatory
proteins,
gntR
family 1.202 1.708
3.357
1.7E
−
4
3.2E
−
06
4.8E
−
11
BN5
1373
Pyruvate
kinase
1.197
1.441
2.208
2.5E
−
05
2.1E
−
06
1.0E
−
08
BN5
2756
Asparagine
synthetase
(asnB)
1.146
1.197
1.963
2.4E
−
05
8.1E
−
06
2.2E
−
08
BN5
0180
Ammonia
permease
(amtB)
1.118
2.213
4.699
6.8E
−
5
2.9E
−
8
4.4E
−
12
BN5
2493
Bacterial
regulatory
protein,
MarR
1.092
2.145
1.411
5.2E
−
4
4.6E
−
07
2.8E
−
05
BN5 1499 Aspartate
kinase −1.078 −1.053 −2.352
2.6E
−
4
2.1E
−
4
3.2E
−
08
BN5
2445
Cytochrome
c
oxidase
cbb3-type
(ccoN3)
−1.111
−1.444
−1.045
2.7E
−
4
1.3E
−
06
2.1E
−
3
BN5 2705 ArsA,
arsenite-activated
ATPase
(arsA)
−1.200
−2.052
−1.069
1.5E
−
05
1.8E
−
08
1.6E
−
05
BN5
0188
PLP-dependent
decarboxylase
(lysA)
−1.270
−1.086
−1.168
9.8E
−
06
3.0E
−
05
7.5E
−
06
BN5
4502
ATPase
F0/V0
complex,
sunibut
C
(atpE)
−1.635
−1.568
−3.1809
7.5E
−
05
9.3E
−
05
2.1E
−
08
BN5
1328
Efflux
transporter
RND-HAE1
(mexD)
−1.730
−1.013
−2.097
1.0E
−
05
1.1E
−
3
3.8E
−
07
BN5
2182
Citrate
synthase
I
(gltA)
−1.774
−1.462
−3.282
1.2E
−
05
4.1E
−
05
4.3E
−
09
BN5 4499 ATPase,
F1
complex,
alpha
subunit
(atpA)
−1.807
−2.160
−3.914
1.2E
−
4
1.2E
−
05
9.8E
−
09
BN5
4497
ATPase,
F1
complex,
beta
subunit
(atpD)
−1.831
−2.277
−4.243
5.8E
−
06
2.6E
−
07
1.7E
−
10
BN5
2444
Cytochrome
c
oxidase
cbb3-type
(ccoP)
−1.972
−3.515
−1.939
9.1E
−
05
1.2E
−
07
4.4E
−
05
BN5
4498
ATPase,
F1
complex,
gamma
subunit
(atpG)
−1.995
−2.488
−4.306
4.2E
−
05
2.4E
−
06
2.6E
−
09
BN5
4500
ATP
synthase
F1,
delta
subunit
(atpH)
−2.114
−2.031
−3.572
4.1E
−
06
4.3E
−
06
3.7E
−
09
BN5
4501
ATPase,
F0
complex,
subunit
B
(atpF)
−2.227
−1.942
−3.819
2.6E
−
06
5.5E
−
06
2.0E
−
09
BN5
4309
Oxaloacetate
decarboxylase
(oadA)
−3.419
−3.827
−3.302
1.4E
−
11
2.8E
−
12
2.9E
−
11
aGene
IDs
refer
to
accession
number
HG916826
(Wibberg
et
al.,
2014).
Among
those
genes
that
increased
their
expression
exclu-
sively
in
sodium
cyanide
it
was
found
the
nitrilase/cyanide
hydratase
nit3
gene
(Table
S3,
Supplementary
material).
Oxaloac-
etate
has
been
described
to
be
essential
in
the
cyanide
degradation
pathway
of
P.
pseudoalcaligenes
CECT5344,
since
is
specifically
produced
by
a
malate:quinone
oxidoreductase
in
response
to
cyanide.
Both
compounds
chemically
react
to
generate
the
oxaloacetate–cyanohydrin,
which
is
further
used
by
the
nitrilase
NitC
that
converts
the
cyanohydrin
into
ammonium
for
its
incorpo-
ration
to
carbon
skeletons
by
the
GS/GOGAT
cycle
(Luque-Almagro
et
al.,
2011c;
Estepa
et
al.,
2012).
Although
the
nitrilase
NitC,
but
not
the
nitrilase
Nit3,
it
is
essential
for
cyanide
assimilation
in
the
strain
CECT5344
(Estepa
et
al.,
2012),
a
residual
nitrilase
activity
that
could
be
attributed
to
the
nitrilase
Nit3
activity
remains
in
a
NitC
defective
mutant
strain
of
P.
pseudoalcaligenes
CECT5344.
However,
the
NitC
mutant
strain
is
unable
to
grow
with
cyanide
as
the
sole
nitrogen
source
(Estepa
et
al.,
2012).
Another
gene
that
increased
its
expression
exclusively
in
sodium
cyanide
(Table
S3,
Supplementary
material)
encodes
a
2-methylcitrate
synthase,
an
acyl-CoA
transferase
that
uses
oxaloacetate
and
propionyl-CoA
as
substrates
to
yield
2-methylcitrate
(Gerike
et
al.,
1998).
As
indi-
cated,
oxaloacetate
is
produced
as
the
first
intermediate
of
the
cyanide
assimilation/degradation
pathway
in
the
strain
CECT5344
(Estepa
et
al.,
2012),
and
probably
other
enzymes
that
use
this
ketoacid
as
substrate
are
also
induced.
On
the
other
hand,
a
large
number
of
genes
encoding
metalloenzymes
were
repressed
by
cyanide
(Table
S3,
Supplementary
material)
and
this
could
be
a
collateral
effect
to
the
enzyme
inhibition
caused
by
cyanide,
which
displays
a
high
affinity
for
the
metallic
centers.
Several
genes
encoding
metal
transporters
have
been
found
induced
in
cells
grown
with
the
jewelry
wastewater
(Table
S4,
Supplemen-
tary
material).
The
Fe3+-siderophore
transporters
are
ABC-type
or
TonB-dependent
systems
for
iron
acquisition,
allowing
cell
sur-
vival
in
the
presence
of
cyanide
(Crosa
and
Walsh,
2002).
The
jewelry
wastewaster
contains
free
and
metal−bound
cyanide,
met-
als
like
iron,
copper
and
zinc,
and
also
small
traces
of
nitrite
and
cyanate
(concentrations
in
the
mM
range).
Metal
extruders
are
induced
as
detoxification
mechanisms
in
the
presence
of
high
concentrations
of
metals.
Bacterial
metal
efflux
pumps
can
be
clas-
sified
in
three
different
types:
the
chemiosmotic
gradient
(H+or
K+)-dependent
cation
diffusion
facilitator
(CDF)
family,
the
efflux
P1-type
ATPases,
and
the
RND
(resistance-nodulation-cell
division)
efflux
pumps
that
are
composed
of
an
outer
membrane
protein,
a
periplasmic
component
with
a
small
membrane
hydrophobic
region
and
an
integral
membrane
component
(Nies,
2003).
Genes
encoding
several
types
of
metal
extruders
have
been
found
induced
in
cells
grown
with
the
jewelry
wastewater,
including
P-type
ATPases
for
Cu+or
Cd2+ extrusion,
multidrug
efflux
pumps
of
the
178
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
Fig.
2.
Validation
by
qPCR
of
the
microarray
data.
q-PCR
analysis
(left
panels)
and
microarrays
data
(right
panels).
The
selected
genes
nitC/nit2
(BN5
1632),
nit3
(BN5
3251),
nit4
(BN5
1912)
and
aguB
(BN5
0258)
encode
proteins
that
belong
to
the
supernitrilase
family
(Podar
et
al.,
2005),
and
the
cioA
(BN5
1902)
and
cioB
(BN5
1903)
genes
code
for
the
cyanide-insensitive
terminal
oxidase.
(A)
Gene
expression
in
cells
grown
under
nitrogen
starvation.
(B)
Gene
expression
in
cells
grown
with
sodium
cyanide.
(C)
Gene
expression
in
cells
grown
with
the
cyanide-containing
wastewater
jewelry
residue.
Gene
expression
in
all
three
nitrogen
conditions
was
referred
to
that
obtained
in
ammonium.
q-PCR
data
represent
an
average
of
triplicate
(±standard
deviation).
RND
family
and
regulatory
genes
involved
in
metal
detoxification
systems.
Additionally,
a
gene
that
codes
for
a
malate:
quinone
oxidoreductase
B
(mqoB)
was
induced
by
the
jewelry
residue.
As
mentioned
above,
a
malate:quinone
oxidoreductase
is
involved
in
the
first
step
of
cyanide
degradation
in
P.
pseudoalcaligenes
CECT5344
by
converting
l-malate
into
oxaloactetate,
and
it
is
also
a
key
enzyme
in
carbon
metabolism
since
this
bacterial
strain
lacks
malate
dehydrogenase
(Luque-Almagro
et
al.,
2011c).
Analy-
sis
of
the
whole
genome
sequence
of
the
strain
CECT53344
reveals
the
existence
of
two-genes
encoding
malate:quinone
oxidoreduc-
tases,
mqoA
(BN5
0860)
and
mqoB
(BN5
1358),
which
share
52%
identity
and
71%
similarity.
The
TargetP
program
(tp://www.cbs.
dtu.dk/services/TargetP/)
has
predicted
unequivocally
a
subcellu-
lar
location
of
MqoA
in
the
cytoplasm
but
an
unclear
subcellular
location
for
the
MqoB
enzyme,
which
could
be
located
either
in
the
cytoplasm
or
in
the
periplasm.
The
mqoB
gene
was
induced
by
the
jewelry
wastewater
whereas
the
mqoA
gene
was
expressed
in
cells
grown
with
all
different
nitrogen
sources
tested
in
this
work.
Therefore,
the
P.
pseudoalcaligenes
CECT5344
MqoA
is
an
essential
key
both
in
central
carbon
metabolism
and
in
the
cyanide
assim-
ilation/detoxification
pathway,
but
function
of
the
mqoB
gene,
which
is
specifically
induced
by
the
cyanide-containing
jewelry
wastewater,
remains
unknown.
Although
a
gene
encoding
an
arse-
nate
reductase
was
also
induced
by
the
jewelry
residue,
arsenic
derivatives
could
not
be
detected
in
this
industrial
waste.
How-
ever,
it
has
been
described
that
other
metals
act
as
inducers
of
the
arsenate
reductase
(Park
and
Ely,
2008).
Specifically
repressed
by
the
jewelry
liquid
residue
were
the
zur
gene
that
codes
for
an
iron
and
zinc
uptake
regulator
and
the
DNA-binding
ferritin-like
dps
gene
(Table
S4,
Supplementary
material).
It
has
been
previously
demonstrated
that
the
ferric-uptake
regulator
family
protein
Zur
represses
genes
involved
in
zinc
uptake
in
Corynebacterium
diphthe-
ria
under
zinc-rich
conditions
(Smith
et
al.,
2009).
The
ferritin-like
Dps
proteins
may
participate
in
oxidative
damage
protection
by
iron-binding,
avoiding
free
radicals
and
reactive
oxygen
species
for-
mation
by
the
Fenton
reaction
(Bellapadrona
et
al.,
2010).
Recently,
it
has
been
postulated
a
role
for
a
Dps
protein
in
copper
homeosta-
sis
in
E.
coli.
Curiously,
in
this
bacterium
the
intracellular
levels
of
copper
decrease
when
the
Dps
protein
is
overexpressed
(Thieme
and
Grass,
2010).
To
investigate
the
role
of
the
Dps
ferrin-like
pro-
tein
in
the
cyanide
assimilation/degradation
process
of
the
strain
CECT5344,
a
Dps
mutant
strain
of
P.
pseudoalcaligenes
CECT5344
has
been
generated.
Growth
of
the
Dps
mutant
was
affected
only
in
the
presence
of
a
high
concentration
of
copper
with
ammonium
as
nitrogen
source,
indicating
a
role
for
Dps
protein
as
copper
chelator.
However,
growth
of
the
Dps
mutant
was
not
affected
in
cyanide-
containing
media
or
in
the
presence
of
high
concentration
of
iron,
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
179
suggesting
that
iron
homeostasis
by
the
Dps
protein
during
cyanide
assimilation/detoxification
does
not
occurs.
The
nitrilase
nit2/nitC
and
nit4
genes,
the
latest
coding
for
a
putative
4-hydroxyalanine
nitrilase,
and
a
malic
enzyme-encoding
gene
were
induced
in
cells
grown
with
both
sodium
cyanide
and
cyanide-containing
jewelry
wastewater
(Table
S5).
The
oxaloac-
etate
cyanohydrin
(a
nitrile)
that
is
produced
during
cyanide
assimilation
in
P.
pseudoalcaligenes
CECT5344
(Estepa
et
al.,
2012)
might
act
as
inducer
of
other
nitrilases
like
Nit4
that
also
use
nitriles
as
substrates.
Thus,
the
nitrilase
Nit4
could
contribute
to
the
residual
activity
found
in
the
NitC
mutant
of
P.
pseudoalcali-
genes
CECT5344,
but
it
is
not
essential
for
cyanide
assimilation
in
the
strain
CECT5344
(Estepa
et
al.,
2012).
Malic
enzyme
catalyzes
oxidative
decarboxylation
of
malate
to
pyruvate
and
this
3-oxoacid
also
reacts
with
free
cyanide
to
produce
a
cyanohydrin
that
can
be
assimilated
through
the
nitrilase
NitC
(Estepa
et
al.,
2012).
This
pro-
cess
may
contribute
to
decrease
the
concentration
of
free
cyanide,
which
is
much
more
toxic
and
reactive
than
its
derivative
organic
forms
(cyanohydrins).
The
cioB
gene
that
codes
for
the
terminal
cbb3-type
oxidase
required
for
cyanide
insensitive
respiration
has
been
also
found
induced
by
both
sodium
cyanide
and
the
jewelry
wastewater
(Table
S5).
Mutational
analysis
of
the
cioB
and
cioA
genes
coding
for
the
two
subunits
of
the
terminal
oxidase
revealed
that
these
genes
are
essential
to
survive
on
cyanide
(Quesada
et
al.,
2007).
Several
genes
located
downstream
cioAB
genes
that
code
for
PLP-dependent
aminotransferases
were
up-regulated
in
media
with
cyanide.
Some
of
these
genes
have
been
demonstrated
to
be
cotranscribed
with
the
cioA
genes,
but
their
role
on
cyanide
metabolism
remains
unknown
(Quesada
et
al.,
2007).
The
nitrilase
nit4
gene
is
also
located
downstream
the
cioAB
genes
in
the
strain
CECT5344
and
might
be
also
co-transcribed
with
the
cioAB
genes.
The
isc
genes
for
Fe-S
cluster
assembly,
the
a
lkyl-hydroperoxide
reductase
ahpC
gene
and
an
fpr
gene
were
also
up-regulated
by
cyanide
(Table
S5).
Alkyl-hydroperoxide
reductase
has
been
pos-
tulated
to
be
involved
in
protection
against
hydrogen
peroxide.
In
pathogenic
Staphylococci,
a
link
between
AhpC
and
Hmp
proteins
has
been
established
since
they
participate
in
global
response
to
oxidative
stress
sharing
common
global
regulators
(Gaupp
et
al.,
2012).
Under
oxidative
stress
conditions
the
Fe-S
clusters
of
many
proteins
are
very
susceptible
to
oxidative
inactivation;
hence,
in
aerobic
bacteria
have
evolved
Fe-S
cluster
repair
mechanisms
like
isc
genes
(Gaupp
et
al.,
2012).
Rhodanases
are
involved
in
detox-
ification
of
cyanide
in
some
microorganisms
since
these
enzymes
catalyze
the
transfer
of
sulfur
to
cyanide
producing
thiocyanate,
a
cyano-derivative
less
toxic
than
cyanide
(Park
and
Ely,
2008).
However,
in
the
strain
CECT5344
the
rhodanase-encoding
gene
was
down-regulated
in
nitrogen-limiting
conditions
but
also
in
the
presence
of
sodium
cyanide
(Table
S6,
Supplementary
material),
suggesting
its
lack
of
functionality
in
cyanide
detoxification
under
these
growth
conditions.
A
large
number
of
genes
up-regulated
by
cyanide
(sodium
cyanide
and
jewelry
wastewater)
can
be
grouped
in
three
different
functional
categories
(GOStats
analysis)
that
include
copper
ion
binding,
terminal
oxidases
(cyoA,
coxAB
and
cox11
genes),
iron–sulfur
cluster
binding
(isc
genes
and
others)
and
pyridoxal
phosphate
binding
(aminotransferases).
This
could
be
related
to
the
cyanide
affinity
for
metals
and
cofactors.
Genes
involved
in
nitrate/nitrite
assimilation
were
induced
in
the
strain
CECT5344
in
response
to
the
jewelry
wastewater
and
nitrogen-limiting
conditions,
both
compared
against
ammonium
(Table
S7,
Supplementary
material).
Genes
involved
in
bacterial
inorganic
nitrogen
(nitrate/nitrite)
assimilation
have
been
widely
described
to
be
induced
under
nitrogen-limiting
conditions
in
a
great
variety
of
Gram
negative
bacteria,
usually
through
the
NtrBC
global
nitrogen
regulatory
system
(Luque-Almagro
et
al.,
2011b).
The
nitrate-assimilating
genes
were
also
induced
in
the
strain
CECT5344
in
response
to
the
jewelry
residue
since
it
contains
small
amounts
of
nitrite
(mM
range)
that
may
act
as
an
inducer.
It
is
worth
nothing
that
the
polyhydrohyalkanoate
structural
and
regulatory
phaI
gene
was
also
up-regulated
in
cells
grown
with
the
jewelry
wastewater
and
under
nitrogen
limiting
conditions.
The
phaI
gene
is
clustered
together
other
genes
involved
in
polyhydroxyalka-
noates
metabolism
in
P.
pseudoalcaligenes
CECT5344,
which
has
been
recently
described
as
a
cyanide-degrading
bacterium
with
by-
product
(polyhydroxyalkanoates)
formation
capacity.
Therefore,
the
strain
CECT5344
could
be
used
in
bioremediation
of
indus-
trial
residues
containing
cyanide,
while
concomitantly
generates
by-products
like
polyhydroxyalkanoates
with
a
biotechnological
added
value
(Manso
et
al.,
2015).
DNA
microarrays
of
P.
pseudoalcaligenes
CECT5344
in
response
to
sodium
cyanide,
the
jewelry
wastewater
and
nitrogen-limiting
conditions,
all
three
compared
against
ammonium,
showed
sev-
eral
up-regulated
genes
(Table
2;
Table
8,
Supplementary
material)
including
the
nitC
gene
that
codes
for
the
nitrilase
NitC
essential
for
cyanide
assimilation
in
the
strain
CECT5344
(Estepa
et
al.,
2012),
the
GCN5-N-acetyltransferase
gene
and
other
genes
of
unknown
function
that
constitute
a
single
transcriptional
unit
with
the
nitC
gene.
Also
induced
in
cells
grown
in
all
three
media
were
genes
coding
for
GntR–MocR
and
pyridoxal
phosphate
(PLP)-dependent
aminotransferases,
biotin
transport
and
synthesis,
and
the
ABC-
type
cyanate
transporter
(cynABD)
and
cyanase
(cynS)
required
for
cyanate
assimilation.
The
GntR–MocR
proteins
are
transcrip-
tional
regulators
containing
a
DNA-binding
HTH
motif
and
an
aminotransferase
domain.
In
MocR-like
proteins,
PLP
is
required
as
cofactor
for
both
aminotransferase
and
regulation
capacities.
The
most
relevant
evidence
comes
from
Streptomyces
venezuelae
PdxR,
which
is
involved
directly
in
the
regulation
of
pyridoxal
phos-
phate
synthesis
(Rigali
et
al.,
2002).
However,
relationship
between
cyanide,
nitrogen
starvation
and
the
coenzymes
PLP
and
biotin
is
still
unkmown.
Cyanate
and
cyanide
assimilation/detoxification
are
separate
pathways
in
P.
pseudoalcaligenes
CECT5344,
since
a
mutant
strain
defective
in
the
cyanase
cynS
gene
uses
cyanide
and
it
is
able
to
grow
with
this
nitrogen
source
at
a
similar
rate
to
that
presented
by
the
wild
type
strain
(Luque-Almagro
et
al.,
2008).
However,
cyanide
and
cyanate
are
indirectly
connected,
as
suggests
by
the
induction
of
the
cyanate
transporter
and
the
cyanase
genes
in
the
cyanide-containing
media.
It
has
been
pos-
tulated
that
in
the
presence
of
cyanide
electrons
blockage
occurs
in
the
respiratory
electron
transfer
chain
as
a
result
of
cyanide-
inhibition
of
terminal
oxidases
and,
as
a
consequence,
free
radicals
and
reactive
oxygen
species
are
produced.
Oxidation
of
cyanide
by
these
reactive
oxygen
species
can
lead
to
the
formation
of
cyanate
(Sarla
et
al.,
2004).
Interestingly,
the
nitrilase
nitC
gene
was
up-
regulated
in
cells
grown
in
cyanide-containing
media
(NaCN
and
CN-WW)
and
also
in
nitrogen
starvation
conditions.
Furthermore,
the
nitC
gene
expression
pattern
was
similar
to
that
shown
by
genes
involved
in
assimilation
of
other
nitrogenous
compounds,
such
the
nas
genes
(nitrate/nitrite
assimilation)
and
the
gln/glt
genes
(ammonia
assimilation),
suggesting
that
the
nitC
gene
functions
as
part
of
an
assimilatory
pathway
to
use
organic
nitriles
as
nitrogen
source
for
growth.
Therefore,
cyanide-assimilation
conditions
may
provoke
nitrogen-limitation
responses,
as
occurs
when
nitrate
and
other
alternative
nitrogen
sources
are
used.
This
finding
has
been
described
in
a
previous
proteomic
analysis
(Luque-Almagro
et
al.,
2007)
and
it
is
also
supported
by
the
present
microarray
study
and
additional
microarray
data
described
in
other
bacteria
(Silberbach
et
al.,
2005),
showing
induction
of
genes
encoding
glutamine
syn-
thetase,
nitrate
reductase
and
ammonium
transport.
Considering
other
nitrilase
genes,
the
nit3
gene
was
induced
exclusively
by
sodium
cyanide
whereas
the
nit1
and
nit4
genes
were
induced
with
sodium
cyanide
and
cyanide-containing
jewelry
wastewater.
The
fact
that
these
nitrilases
genes
(nit1,
nit3
and
nit4)
were
not
induced
in
P.
pseudoalcaligenes
CECT5344
cells
grown
under
nitrogen
180
V.M.
Luque-Almagro
et
al.
/
Journal
of
Biotechnology
214
(2015)
171–181
starvation
conditions,
in
contrast
to
the
nitC
gene,
suggests
a
role
for
these
three
nitrilases
in
detoxification
of
cyanide
rather
than
in
assimilation
of
this
nitrogenous
compound.
Although
studying
the
neighborhood
of
a
specific
gene
can
be
useful
to
predict
a
possi-
ble
gene
function,
this
is
not
the
case
of
the
nitrilases
nit1
and
nit3
since
they
display
surrounding
genes
of
unknown
function.
How-
ever,
the
nitrilase
nit4
gene
is
clustered
together
other
genes
that
were
induced
by
sodium
cyanide
and
the
jewelry
residue,
such
as
the
cioB
gene
for
cyanide-insensitive
respiration
(Wibberg
et
al.,
2014).
The
aguB
gene
(BN5
0258)
also
codes
for
a
member
of
the
nitrilase/C–N
hydrolase
superfamily
that
is
not
considered
a
proper
nitrilase
(Podar
et
al.,
2005).
This
gene
was
specifically
repressed
in
cells
grown
with
the
jewelry
wastewater.
It
is
worth
nothing
that
these
five
putative
nitrilase–superfamily
proteins
of
P.
pseudoal-
caligenes
CECT5344
do
not
share
significant
sequence
homology
(Luque-Almagro
et
al.,
2013).
5.
Concluding
remarks
DNA
microarrays
of
P.
pseudoalcaligenes
CECT5344
in
response
to
cyanide
confirmed
the
essential
role
of
several
genes
previ-
ously
established
by
mutational
analysis,
including
the
nitrilase
nitC-encoding
gene
for
cyanide
assimilation,
and
the
cioAB
genes
for
cyanide
insensitive
respiration.
The
induction
by
cyanide
of
these
genes,
which
are
essential
for
the
strain
CECT5344
to
sur-
vive
in
the
presence
of
cyanide,
validates
the
DNA
microarrays
data
presented
in
this
work.
Furthermore,
under
nitrogen
starva-
tion
conditions
were
induced
several
genes
like
glnK,
ntrB
and
amtB
previously
described
in
different
bacteria
to
be
induced
when
the
nitrogen
concentration
in
the
media
is
scarce.
DNA
microarray
data
have
been
also
validated
by
qPCR
analysis
of
four
nitrilases
genes
differentially
expressed
in
P.
pseudoalcaligenes
CECT5344
accord-
ing
to
DNA
microarrays.
Several
genes
up-regulated
by
cyanide
(sodium
cyanide
and
the
cyanide-containing
jewelry
wastewater)
might
have
a
relevant
role
in
cyanide
assimilation/detoxification,
which
will
require
future
work
in
order
to
optimize
bioremedia-
tion
of
industrial
wastes
containing
high
concentration
of
cyanide.
It
is
worth
highlight
that
is
of
special
interest
to
further
investi-
gate
the
link
between
cyanide
degradation
and
carbon
metabolism,
especially
with
polyhydroxyalkanoates
production.
The
relevance
of
metal
extrusion
systems
in
detoxification
of
the
jewelry
wastew-
ater
has
been
also
revealed
in
this
study.
Acknowledgements
This
work
was
funded
by
Ministerio
de
Economía
y
Competi-
tividad
(Grant
BIO2011-30026-C02-02)
and
by
Junta
de
Andalucía
(Grant
CVI-7560).
We
also
thank
FCC-Ámbito,
SAVECO,
AVENIR
and
MAGTEL
for
fruitful
collaborations.
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.jbiotec.2015.09.
032.
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