24 research outputs found
Cyanate Assimilation by the Alkaliphilic Cyanide-Degrading Bacterium Pseudomonas pseudoalcaligenes CECT5344: Mutational Analysis of the cyn Gene Cluster
The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 can grow with cyanate, cyanide, or cyanide-containing industrial residues as the sole nitrogen source, but the assimilation of cyanide and cyanate takes place through independent pathways. Therefore, cyanide degradation involves a chemical reaction between cyanide and oxaloacetate to form a nitrile that is hydrolyzed to ammonium by the nitrilase NitC, whereas cyanate assimilation requires a cyanase that catalyzes cyanate decomposition to ammonium and carbon dioxide. The P. pseudoalcaligenes CECT5344 cynFABDS gene cluster codes for the putative transcriptional regulator CynF, the ABC-type cyanate transporter CynABD, and the cyanase CynS. In this study, transcriptional analysis revealed that the structural cynABDS genes constitute a single transcriptional unit, which was induced by cyanate and repressed by ammonium. Mutational characterization of the cyn genes indicated that CynF was essential for cynABDS gene expression and that nitrate/nitrite transporters may be involved in cyanate uptake, in addition to the CynABD transport system. Biodegradation of hazardous jewelry wastewater containing high amounts of cyanide and metals was achieved in a batch reactor operating at an alkaline pH after chemical treatment with hydrogen peroxide to oxidize cyanide to cyanate
Exploring anaerobic environments for cyanide and cyano-derivatives microbial degradation
Cyanide is one of the most toxic chemicals for living organisms described so far. Its toxicity is mainly based on the high affinity
that cyanide presents toward metals, provoking inhibition of essential metalloenzymes. Cyanide and its cyano-derivatives are
produced in a large scale by many industrial activities related to recovering of precious metals in mining and jewelry, coke
production, steel hardening, synthesis of organic chemicals, and food processing industries. As consequence, cyanide-containing
wastes are accumulated in the environment becoming a risk to human health and ecosystems. Cyanide and related compounds,
like nitriles and thiocyanate, are degraded aerobically by numerous bacteria, and therefore, biodegradation has been offered as a
clean and cheap strategy to deal with these industrial wastes. Anaerobic biological treatments are often preferred options for
wastewater biodegradation. However, at present very little is known about anaerobic degradation of these hazardous compounds.
This review is focused on microbial degradation of cyanide and related compounds under anaerobiosis, exploring their potential
application in bioremediation of industrial cyanide-containing wastes
Assimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344: from omic approaches to biotechnological applications
Mining, jewellery and metal-processing industries use cyanide for extracting gold and other valuable metals, generating large amounts of highly toxic wastewater. Biological treatments may be a clean alternative under the environmental point of view to the conventional physical or chemical processes used to remove cyanide and related compounds from these industrial effluents. Pseudomonas pseudoalcaligenes CECT5344 can grow under alkaline conditions using cyanide, cyanate or different nitriles as the sole nitrogen source, and is able to remove up to 12 mM total cyanide from a jewellery industry wastewater that contains cyanide free and complexed to metals. Complete genome sequencing of this bacterium has allowed the application of transcriptomic and proteomic techniques, providing a holistic view of the cyanide biodegradation process. The complex response to cyanide by the cyanotrophic bacterium P. pseudoalcaligenes CECT5344 and the potential biotechnological applications of this model organism in the bioremediation of cyanide-containing industrial residues are reviewed
Alternative pathway for 3-cyanoalanine assimilation in pseudomonas pseudoalcaligenes CECT5344 under Noncyanotrophic conditions
3-Cyanoalanine and cyanohydrins are intermediate nitriles produced in cyanide degradation pathways in plants and bacteria. 3-Cyanoalanine is generated from cyanide by the 3-cyanoalanine synthase, an enzyme mainly characterized in cyanogenic plants. NIT4-type nitrilases use 3-cyanoalanine as a substrate, forming ammonium and aspartate. In some organisms, this enzyme also generates asparagine through an additional nitrile hydratase activity. The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 assimilates cyanide through an intermediate cyanohydrin, which is further converted into ammonium by the nitrilase NitC.
This bacterium also contains three additional nitrilases, including Nit4. In this work, a proteomic analysis of P. pseudoalcaligenes CECT5344 cells grown with 3-cyanoalanine as the sole nitrogen source has revealed the overproduction of different proteins involved in nitrogen metabolism, including the nitrilase NitC. In contrast, the nitrilase Nit4 was not induced by 3-cyanoalanine, and it was only overproduced in cells grown with a cyanide-containing jewelry-manufacturing residue. Phenotypes of single and double mutant strains defective in nit4 or/and nitC revealed the implication of the nitrilase NitC in the assimilation of 3-cyanoalanine and suggest that the 3-cyanoalanine assimilation pathway in P. pseudoalcaligenes CECT5344 depends on the presence or absence of cyanide. When cyanide is present, 3-cyanoalanine is assimilated via Nit4, but in the absence of cyanide, a novel pathway for 3-cyanoalanine assimilation, in which the nitrilase NitC uses the nitrile generated after deamination of the a-amino group from 3-cyanoalanine, is proposed
Quantitative proteomic analysis of Pseudomonas pseudoalcaligenes CECT5344 in response to industrial cyanide-contain ing wastewaters using Liquid Chromatography- Mass Spectrometry/Mass Spectrometry (LC- MS/MS)
Biological
treatments
to degrade
cyanide
are
a powerful
technology
for
cyanide
removal
from
industrial
wastewaters.
It has
been
previously
demonstrated
that
the
alkaliphilic
bacterium
Pseudomonas
pseudoalcali
genes
CECT5344
is able
to use
free
cyanide
and
several
metal
−
cyanide
complexes
as
the
sole
nitrogen
source.
In this
work,
the
strain
CECT5344
has
been
used
for
detoxification
of the
different
chemical
forms
of cyanide
that
are
present
in alkaline
wastewaters
from
the
jewelry
industry.
This
liquid
residue
also
contains
large
concentration
s
of metals
like
iron,
copper
and
zinc,
making
this
wastewater
even
more
toxic.
To
elucidate
the
molecular
mechanisms
involved
in the
bioremediation
process,
a quantitative
proteomic
anal-
ysis
by
LC-MS/MS
has
been
carried
out
in
P
.
pseudoalcaligene
s
CECT5344
cells
grown
with
the
jewelry
residue
as
sole
nitrogen
source.
Different
proteins
related
to cyanide
and
cyanate
assimilation,
as
well
as
other
proteins
involved
in transport
and
resistance
to metals
were
induced
by
the
cyanide-cont
aining
jewelry
residue.
GntR-like
regulatory
proteins
were
also
induced
by
this
industrial
residue
and
mutational
analysis
revealed
that
GntR-like
regulatory
proteins
may
play
a role
in the
regulation
of cyanide
assimilation
in
P
.
pseudoalcaligene
s
CECT5344.
The
strain
CECT5344
has
been
used
in a batch
reactor
to remove
at pH
9 the
dif-
ferent
forms
of cyanide
present
in industrial
wastewaters
from
the
jewelry
industry
(0.3
g/L,
ca
.
12
mM
total
cyanide,
including
both
free
cyanide
and
metal
−
cyanide
complexes).
This
is
the
first
report
describing
the
biological
removal
at alkaline
pH
of such
as
elevated
concentra-
tion
of cyanide
present
in a heterogeneou
s mixture
from
an
industrial
source
Role of the dihydrodipicolinate synthase DapA1 on iron homeostasis during cyanide assimilation by the alkaliphilic bacterium pseudomonas pseudoalcaligenes CECT5344
Cyanide is a toxic compound widely used in mining and jewelry industries, as well as in the synthesis of many different chemicals. Cyanide toxicity derives from its high affinity for metals, which causes inhibition of relevant metalloenzymes. However, some cyanide-degrading microorganisms like the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 may detoxify hazardous industrial wastewaters that contain elevated cyanide and metal concentrations. Considering that iron availability is strongly reduced in the presence of cyanide, mechanisms for iron homeostasis should be required for cyanide biodegradation. Previous omic studies revealed that in the presence of a cyanide-containing jewelry residue the strain CECT5344 overproduced the dihydrodipicolinate synthase DapA1, a protein involved in lysine metabolism that also participates in the synthesis of dipicolinates, which are excellent metal chelators. In this work, a dapA1– mutant of P. pseudoalcaligenes CECT5344 has been generated and characterized. This mutant showed reduced growth and cyanide consumption in media with the cyanide-containing wastewater. Intracellular levels of metals like iron, copper and zinc were increased in the dapA1– mutant, especially in cells grown with the jewelry residue. In addition, a differential quantitative proteomic analysis by LC-MS/MS was carried out between the wild-type and the dapA1– mutant strains in media with jewelry residue. The mutation in the dapA1 gene altered the expression of several proteins related to urea cycle and metabolism of arginine and other amino acids. Additionally, the dapA1– mutant showed increased levels of the global nitrogen regulator PII and the glutamine synthetase. This proteomic study has also highlighted that the DapA1 protein is relevant for cyanide resistance, oxidative stress and iron homeostasis response, which is mediated by the ferric uptake regulator Fur. DapA1 is required to produce dipicolinates that could act as iron chelators, conferring protection against oxidative stress and allowing the regeneration of Fe-S centers to reactivate cyanide-damaged metalloproteins
The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions
Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins
Holistic view of biological nitrogen fixation and phosphorus mobilization in Azotobacter chroococcum NCIMB 8003
Nitrogen (N) and phosphorus (P) deficiencies are two of the most agronomic
problems that cause significant decrease in crop yield and quality. N and P chemical
fertilizers are widely used in current agriculture, causing environmental problems and
increasing production costs. Therefore, the development of alternative strategies to
reduce the use of chemical fertilizers while maintaining N and P inputs are being
investigated. Although dinitrogen is an abundant gas in the atmosphere, it requires
biological nitrogen fixation (BNF) to be transformed into ammonium, a nitrogen
source assimilable by living organisms. This process is bioenergetically expensive
and, therefore, highly regulated. Factors like availability of other essential elements,
as phosphorus, strongly influence BNF. However, the molecular mechanisms of
these interactions are unclear. In this work, a physiological characterization of BNF
and phosphorus mobilization (PM) from an insoluble form (Ca3(PO4)2) in Azotobacter
chroococcum NCIMB 8003 was carried out. These processes were analyzed
by quantitative proteomics in order to detect their molecular requirements and
interactions. BNF led to a metabolic change beyond the proteins strictly necessary
to carry out the process, including the metabolism related to other elements, like
phosphorus. Also, changes in cell mobility, heme group synthesis and oxidative stress
responses were observed. This study also revealed two phosphatases that seem
to have the main role in PM, an exopolyphosphatase and a non-specific alkaline
phosphatase PhoX. When both BNF and PM processes take place simultaneously, the
synthesis of nitrogenous bases and L-methionine were also affected. Thus, although
the interdependence is still unknown, possible biotechnological applications of these
processes should take into account the indicated factors.Datos de investigación disponibles en: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1129721/full#supplementary-materia
Pseudomonas pseudoalcaligenes CECT5344, a cyanide‑degrading bacterium with by‑product (polyhydroxyalkanoates) formation capacity
Background: Cyanide is one of the most toxic chemicals produced by anthropogenic activities like mining and
jewelry industries, which generate wastewater residues with high concentrations of this compound. Pseudomonas
pseudoalcaligenes CECT5344 is a model microorganism to be used in detoxification of industrial wastewaters containing
not only free cyanide (CN−) but also cyano-derivatives, such as cyanate, nitriles and metal-cyanide complexes.
Previous in silico analyses suggested the existence of genes putatively involved in metabolism of short chain length
(scl-) and medium chain length (mcl-) polyhydroxyalkanoates (PHAs) located in three different clusters in the genome
of this bacterium. PHAs are polyesters considered as an alternative of petroleum-based plastics. Strategies to optimize
the bioremediation process in terms of reducing the cost of the production medium are required.
Results: In this work, a biological treatment of the jewelry industry cyanide-rich wastewater coupled to PHAs production
as by-product has been considered. The functionality of the pha genes from P. pseudoalcaligenes CECT5344
has been demonstrated. Mutant strains defective in each proposed PHA synthases coding genes (Mpha−, deleted in
putative mcl-PHA synthases; Spha−, deleted in the putative scl-PHA synthase) were generated. The accumulation and
monomer composition of scl- or mcl-PHAs in wild type and mutant strains were confirmed by gas chromatographymass
spectrometry (GC–MS). The production of PHAs as by-product while degrading cyanide from the jewelry industry
wastewater was analyzed in batch reactor in each strain. The wild type and the mutant strains grew at similar rates
when using octanoate as the carbon source and cyanide as the sole nitrogen source. When cyanide was depleted
from the medium, both scl-PHAs and mcl-PHAs were detected in the wild-type strain, whereas scl-PHAs or mcl-PHAs
were accumulated in Mpha− and Spha−, respectively. The scl-PHAs were identified as homopolymers of 3-hydroxybutyrate
and the mcl-PHAs were composed of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers.
Conclusions: These results demonstrated, as proof of concept, that talented strains such as P. pseudoalcaligenes
might be applied in bioremediation of industrial residues containing cyanide, while concomitantly generate by-products
like polyhydroxyalkanoates. A customized optimization of the target bioremediation process is required to gain
benefits of this type of approaches