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Cyanide metabolism of Pseudomonas
pseudoalcaligenes CECT5344: role of siderophores
M.-J. Huertas*, V.M. Luque-Almagro*, M. Martı́nez-Luque*, R. Blasco†, C. Moreno-Vivián*, F. Castillo*
and M.-D. Roldán*1
*Departamento de Bioquı́mica y Biologı́a Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071 Córdoba, Spain,
and †Departamento de Bioquı́mica, Biologı́a Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, 11071 Cáceres, Spain
Abstract
Cyanide is one of the most potent and toxic chemicals produced by industry. The jewellery industry of
Córdoba (Spain) generates a wastewater (residue) that contains free cyanide, as well as large amounts
of cyano–metal complexes. Cyanide is highly toxic to living systems because it forms very stable complexes
with transition metals that are essential for protein function. In spite of its extreme toxicity, some organisms
have acquired mechanisms to avoid cyanide poisoning. The biological assimilation of cyanide needs the
concurrence of three separate processes: (i) a cyanide-insensitive respiratory chain, (ii) a system for iron
acquisition (siderophores) and (iii) a cyanide assimilation pathway. Siderophores are low-molecular-mass
compounds (600–1500 Da) that scavenge iron (Fe3+ ) ions (usually with extremely high affinity) from
the environment under iron-limiting conditions. There are two main classes of siderophores: catechol
and hydroxamate types. The catechol-type siderophores chelate ferric ion via a hydroxy group, whereas
the hydroxamate-type siderophores bind iron via a carbonyl group with the adjacent nitrogen. In the
presence of cyanide, bacterial proliferation requires this specific metal uptake system because siderophores
are able to break down cyano–metal complexes. Pseudomonas pseudoalcaligenes CECT5344 is able to use
free cyanide or cyano–metal complexes as nitrogen source. A proteomic approach was used for the isolation
and identification, in this strain, of a protein that was induced in the presence of cyanide, namely CN0, that
is involved in siderophore biosynthesis in response to cyanide. An overview of bacterial cyanide degradation
pathways and the involvement of siderophores in this process are presented.
Introduction
Cyanide is a toxic nitrogen compound for almost all organisms since it binds irreversibly to metalloproteins, such as
the cytochromes involved in all known respiratory processes.
In nature, there are many plants, bacteria, algae and fungi
which are able to produce cyanide. However, contamination
problems are mainly related to human activities. In this
sense, mining, electroplating and jewellery industries generate
effluents with high concentrations of cyanide. Thus the
jewellery industry of Córdoba (Spain) generates a cyanidecontaining wastewater (residue) with approx. 20 g/l free
cyanide (2 ml of this residue is lethal to an adult human) [1].
In addition to free cyanide, this residue contains cyano–metal
complexes, making it even more poisonous. In spite of cyanide toxicity, there are organisms able to survive in its presence and some of them are able to use it as a nitrogen source
[2]. At present, physicochemical treatments are available for
these residues, but they are expensive and also present some
collateral effects. Since cyanide is a natural biodegradable
compound, biological treatments are better indicated to eliminate it from industrial effluents [3]. In the cyanide molecule,
the oxidation state of C (+2, like that in CO) and N (–3,
Key words: alkalophile, biodegradation, cyanide, jewellery residue, Pseudomonas, siderophore.
1
To whom correspondence should be addressed (email [email protected]).
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like that in NH4 + ) makes this compound a bad C source
but a good N source for bacterial growth. Nevertheless,
micro-organisms can metabolize cyanide only when, in addition to a biodegradable pathway to convert cyanide into
an assimilative product (NH4 + ), they also contain a cyanideresistance mechanism (generally an alternative, cyanide-insensitive oxidase) and a system for taking up Fe3+ from the
medium (siderophores), since Fe3+ forms very stable complexes with cyanide and it is not available for the organisms
in the presence of cyanide. Several metabolic pathways have
been described for cyanide biodegradation, and treatments
to eliminate cyanide from contaminated effluents are based
on activated sludges or pure enzymes [2,4]. Unfortunately,
these systems are not useful for complex mixtures of cyanide
and metals. Some phytopathogenic fungi, like Fusarium solani
[5], are able to degrade cyanide, but bacterial biodegradation
shows considerable advantages since bacteria are more easily
manipulated both at biochemical and genetic levels.
Cyanide degradation pathways
Several enzymatic pathways have been described for cyanide
degradation [2–6]. There are three types of enzymatic
reactions of cyanide: substitution/transfer, hydrolytic and
oxidative reactions (Table 1).
The 11th Nitrogen Cycle Meeting 2005
Table 1 Cyanide degradation pathways
Reaction and enzyme
Micro-organism
Oxidative
Cyanide mono-oxygenase
HCN + O2 + H+ + NADPH → HOCN + NADP+ + H2 O
Pseudomonas sp.
Cyanide dioxygenase
HCN + O2 + H+ + NADPH → CO2 + NH3 + NADP+
Cyanase
Pseudomonas fluorescens, Bacillus cereus, Bacillus pumillus
HOCN + HCO3 − + H2 O → 2CO2 + NH3 + OH−
Hydrolytic
Cyanide hydratase
HCN + H2 O → HCONH2
Nitrile hydratase
R-CN + H2 O → RCONH2
Cyanidase
HCN + 2H2 O → HCOOH + NH3
Nitrilase
R-CN + 2H2 O → RCOOH + NH3
Substitution/transfer
Rhodanese
HCN + S2 O3 2− → HCNS + SO3 2−
Cyanoalanine synthase
E. coli, Rhodococcus rhodochrous
Pathogenic fungi
Pseudomonas, Corynebacterium, Brevibacterium
Alcaligenes xylosoxidans
Klebsiella ozaenae, Arthrobacter sp., Pseudomonas aeruginosa, Nocardia sp.
Thiobacillus denitrificans, Bacillus subtilis, Bacillus stearothermophilus
Bacillus megaterium
Cys + HCN → β-cyanoalanine + HS−
Cyanide has a high affinity for sulphur, particularly in
the persulphide form. In this sense, there are two sulphur
transferases able to produce thiocyanate from cyanide. The
physiological function of the rhodanese seems to be the maintenance of the sulphane sulphur pool in organisms and the
incorporation of reduced sulphur for iron/sulphur proteins.
Kinetics studies suggest that the enzyme works in two steps:
first, thiosulphate donates a sulphur to a cysteine thiol on the
protein to form an intermediate, and secondly, cyanide attacks
to produce thiocyanate and regenerate the enzyme (Table 1).
The pyridoxal phosphate enzymes produce nitriles or
α-amino acids from cyanide through substitution reactions.
Among them, the β-cyanoalanine synthase catalyses the substitution of a three-carbon amino acid with cyanide. The
three-carbon substrate is often cysteine or O-acetylserine
(Table 1) [4]. The hydrolytic reactions are mainly characterized for the direct formation of the products, formamide
or formic acid and ammonium, which are less toxic than
cyanide and may also serve for growth. The cyanide hydratase, which produces formamide, has been found in phytopathogenic fungi, although the mechanism of action of this
enzyme remains uncertain since attempts to purify it have
been unsuccessful. Direct hydrolysis of cyanide to formic acid
and ammonium has been demonstrated, and in parallel with
the nitrile-hydrolysing enzyme nitrilase, both have been
called cyanidases (Table 1). The oxidative pathways for
cyanide degradation are unusual since many oxidoreductases
are metalloenzymes which are inhibited by cyanide [4].
However, there are two types of enzymes able to produce
cyanate (cyanide mono-oxygenase) or carbon dioxide and
ammonium (cyanide dioxygenase). Once cyanate is produced, this compound can be transformed into carbon dioxide and ammonium by a cyanase activity (Table 1). Most
cyanotrophic micro-organisms are able to degrade cyanide at
a neutral pH, but under this condition a high concentration
of cyanide evaporates as hydrocyanic acid, a weak acid with
a pK a value of 9.2. Thus it is very important to isolate
cyanotrophic micro-organisms that work at alkaline pH. In
this sense, our research group has isolated an autochthonous
bacterium from the Guadalquivir River in Córdoba, which
is able to degrade free cyanide and cyano–metal complexes
under alkaline conditions (up to pH 12). This bacterium was
identified on the basis of its 16 S rRNA as Pseudomonas
pseudoalcaligenes and deposited in the Colección Española
de Cultivos Tipo as strain 5344. In addition, this strain
tolerates high cyanide concentrations (up to 30 mM) and
uses several nitrogen sources, such as nitrate, nitrite,
cyanate, β-cyanoalanine, cyanoacetamide, nitroferricyanide
(nitroprusside) and cyano–metal complexes [7,8]. All these
characteristics make this strain a model organism to be used
in bioremediation processes and biotreatment of industrial
residues containing cyanide and its derivatives.
Siderophores and bacterial cyanide
degradation
Iron is absolutely required for many biological systems due
to its diverse role in redox reactions, structural functions and
even changing reactivity of the active site residues. Although
iron is the most abundant transition metal, its solubility is
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Figure 1 Types of siderophores
(A) Catechol-type and (B) hydroxamate-type.
very poor. Thus the availability of iron to living systems is
severely limited. In bacteria, iron must be extracted from
the environment or the host by specialized uptake mechanisms. Once iron has been transported, it must be stored
because it presents a high reactivity, producing radicals
that can damage different biomolecules inside the cell [9].
Most of the iron in the biological fluids of vertebrates is
found bound by transferrin, lactoferrin and blood-haem
proteins. A key feature that enables pathogenic bacteria to
survive in a host is the production of siderophores, which
are iron-sequestering compounds [10]. Ferric siderophores
chelate iron from transferrin or lactoferrin or other host proteins and are transported through outer membrane receptors.
These receptors can be classified into two general types depending on the structure of the siderophore molecule: the
catecholate and the hydroxamate types (Figure 1) [9].
Usually, the catecholate siderophores are fluorescents,
whereas the hydroxamate siderophores are non-fluorescents. However, the structure of siderophores is variable
and the only feature that they share is the presence of functional groups that can provide a high-affinity set of ligands,
which are generally oxygenated, for co-ordination of ferric
ions. In addition, there are many others compounds which
combine these types of siderophores or present other chelating compounds such as hydroxyacids. Thus, in Escherichia
coli and Salmonella typhimurium, the 669 Da catecholate
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siderophore enterobactin has been isolated. Ferrichrome is
a 740 Da hydroxamate-type siderophore produced by E. coli
and fungi. Pyoverdins are the most frequent siderophores
produced by Pseudomonas strains and they are fluorescent
[11]. The biosynthetic pathways of siderophores are diverse.
For example, the adjacent hydroxyls of catechol rings are
usually derived from 2,3-dihydroxybenzoate and L-serine,
as represented in enterobactin or the chromophore from
pyoverdins, which seem to have phenylalanine and/or tyrosine as precursors [11]. Often siderophores are transported
to the cytoplasm through an ATP-dependent ABC-type
transporter [12]. The representation of the uptake of Fe(III)ferrichrome transport is shown (Figure 2) as an example
of ferri-siderophore transport in Gram-negative bacteria.
After ferrichrome binds to the receptor, FhuA interacts
with the periplasmic domain of TonB, facilitating the protonmotive-force-driven active transport of the ferrichrome to
the periplasm, where the ferri-siderophore binds to FhuD,
which binds a wide range of hydroxamates and which then
interacts with the cytoplasmic permease FhuBC. In the
cytoplasm, the complex is reduced and the ferrous ion is
removed and chelated by an acceptor molecule [12]. In the
case of cyanotrophic organisms, production of siderophores
is also required to chelate iron since cyanide binds it strongly.
A proteomic analysis of Ps. pseudoalcaligenes CECT5344
cells grown with cyanide reveals the presence of the
The 11th Nitrogen Cycle Meeting 2005
Figure 2 Uptake of ferri-siderophore in Gram-negative
bacteria
The transport of Fe(III)-ferrichrome is shown as an example.
V.M. Luque-Almagro, M. Martı́nez-Luque, R. Blasco, F.
Castillo, C. Moreno-Vivián and M.D. Roldán, unpublished
work).
This work was funded by Ministerio de Ciencia y Tecnologı́a (grant
BMC 2002-04126-CO3-03) and Junta de Andalucı́a (grant CVI-0117).
V.M.L.-A. was a recipient of a fellowship from the Ministerio de
Ciencia y Tecnologı́a and M.-D.R. holds a postdoctoral contract from
Junta de Andalucı́a, Spain.
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protein CN0, and fragments generated by MALDI–TOF-MS
(matrix-assisted laser-desorption ionization–time-of-flight
MS) indicated that it is a formyltransferase probably involved
in pyoverdin-type siderophore biosynthesis (PvdF) (M.J.
Huertas, V.M. Luque-Almagro, M. Martı́nez-Luque, R.
Blasco, F. Castillo, C. Moreno-Vivián and M.D. Roldán, unpublished work). In addition, the strain RC5 of Ps. pseudoalcaligenes CECT5344, which was obtained by Tn5 insertion
mutagenesis, tolerates higher concentrations of cyanide than
the wild-type. Siderophore production analysis reveals
that the RC5 strain is able to produce a siderophore in
response to cyanide which scavenges iron more efficiently
than the one produced by the wild-type strain (M.J. Huertas,
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