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

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

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

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

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

Bacterial nitrate assimilation: gene distribution and regulation

In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS. The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH–nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed

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