Bacterial tolerance and detoxification of cyanide, arsenic and heavy metals: Holistic approaches applied to bioremediation of industrial complex wastes.

Cyanide is a highly toxic compound that is found in wastewaters generated from different industrial activities, such as mining or jewellery. These residues usually contain high concentrations of other toxic pollutants like arsenic and heavy metals that may form different complexes with cyanide. To develop bioremediation strategies, it is necessary to know the metabolic processes involved in the tolerance and detoxification of these pollutants, but most of the current studies are focused on the characterization of the microbial responses to each one of these environmental hazards individually, and the effect of co-contaminated wastes on microbial metabolism has been hardly addressed. This work summarizes the main strategies developed by bacteria to alleviate the effects of cyanide, arsenic and heavy metals, analysing interactions among these toxic chemicals. Additionally, it is discussed the role of systems biology and synthetic biology as tools for the development of bioremediation strategies of complex industrial wastes and co-contaminated sites, emphasizing the importance and progress derived from meta-omic studies.

Quantitative Proteomic Analysis of Cyanide and Mercury Detoxification by Pseudomonas pseudoalcaligenes CECT 5344.

The cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT 5344 uses cyanide and different metal-cyanide complexes as the sole nitrogen source. Under cyanotrophic conditions, this strain was able to grow with up to 100 µM mercury, which was accumulated intracellularly. A quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been applied to unravel the molecular basis of the detoxification of both cyanide and mercury by the strain CECT 5344, highlighting the relevance of the cyanide-insensitive alternative oxidase CioAB and the nitrilase NitC in the tolerance and assimilation of cyanide, independently of the presence or absence of mercury. Proteins overrepresented in the presence of cyanide and mercury included mercury transporters, mercuric reductase MerA, transcriptional regulator MerD, arsenate reductase and arsenical resistance proteins, thioredoxin reductase, glutathione S-transferase, proteins related to aliphatic sulfonates metabolism and sulfate transport, hemin import transporter, and phosphate starvation induced protein PhoH, among others. A transcriptional study revealed that from the six putative merR genes present in the genome of the strain CECT 5344 that could be involved in the regulation of mercury resistance/detoxification, only the merR2 gene was significantly induced by mercury under cyanotrophic conditions. A bioinformatic analysis allowed the identification of putative MerR2 binding sites in the promoter regions of the regulatory genes merR5, merR6, arsR, and phoR, and also upstream from the structural genes encoding glutathione S-transferase (fosA and yghU), dithiol oxidoreductase (dsbA), metal resistance chaperone (cpxP), and amino acid/peptide extruder involved in quorum sensing (virD), among others. IMPORTANCE Cyanide, mercury, and arsenic are considered very toxic chemicals that are present in nature as cocontaminants in the liquid residues generated by different industrial activities like mining. Considering the huge amounts of toxic cyanide- and mercury-containing wastes generated at a large scale and the high biotechnological potential of P. pseudoalcaligenes CECT 5344 in the detoxification of cyanide present in these industrial wastes, in this work, proteomic, transcriptional, and bioinformatic approaches were used to characterize the molecular response of this bacterium to cyanide and mercury, highlighting the mechanisms involved in the simultaneous detoxification of both compounds. The results generated could be applied for developing bioremediation strategies to detoxify wastes cocontaminated with cyanide, mercury, and arsenic, such as those generated at a large scale in the mining industry.

Proteomic Analysis of Arsenic Resistance during Cyanide Assimilation by Pseudomonas pseudoalcaligenes CECT 5344.

Wastewater from mining and other industries usually contains arsenic and cyanide, two highly toxic pollutants, thereby creating the need to develop bioremediation strategies. Here, molecular mechanisms triggered by the simultaneous presence of cyanide and arsenite were analyzed by quantitative proteomics, complemented with qRT-PCR analysis and determination of analytes in the cyanide-assimilating bacterium Pseudomonas pseudoalcaligenes CECT 5344. Several proteins encoded by two ars gene clusters and other Ars-related proteins were up-regulated by arsenite, even during cyanide assimilation. Although some proteins encoded by the cio gene cluster responsible for cyanide-insensitive respiration decreased in the presence of arsenite, the nitrilase NitC required for cyanide assimilation was unaffected, thus allowing bacterial growth with cyanide and arsenic. Two complementary As-resistance mechanisms were developed in this bacterium, the extrusion of As(III) and its extracellular sequestration in biofilm, whose synthesis increased in the presence of arsenite, and the formation of organoarsenicals such as arseno-phosphoglycerate and methyl-As. Tetrahydrofolate metabolism was also stimulated by arsenite. In addition, the ArsH2 protein increased in the presence of arsenite or cyanide, suggesting its role in the protection from oxidative stress caused by both toxics. These results could be useful for the development of bioremediation strategies for industrial wastes co-contaminated with cyanide and arsenic.

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.

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 α-amino group from 3-cyanoalanine, is proposed. IMPORTANCE Nitriles are organic cyanides with important industrial applications, but they are also found in nature. 3-Cyanoalanine is synthesized by plants and some bacteria to detoxify cyanide from endogenous or exogenous sources, but this nitrile may be also involved in other processes such as stress tolerance, nitrogen and sulfur metabolism, and signaling. The cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344 grows with 3-cyanoalanine as the sole nitrogen source, but it does not use this nitrile as an intermediate in the cyanide assimilation pathway. In this work, a quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to study, for the first time, the response to 3-cyanoalanine at the proteomic level. Proteomic data, together with phenotypes of different nitrilase-defective mutants of P. pseudoalcaligenes CECT5344, provide evidence that in the absence of cyanide, the nitrilase Nit4 is not involved in 3-cyanoalanine assimilation, and instead, the nitrilase NitC participates in a novel alternative 3-cyanoalanine assimilation pathway.

Bioremediation of cyanide-containing wastes: The potential of systems and synthetic biology for cleaning up the toxic leftovers from mining.

Synthetic biology could harness the ability of microorganisms to use highly toxic cyanide compounds for growth applied to bioremediation of cyanide-contaminated mining wastes and areas.

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.

Quantitative proteomic analysis of Pseudomonas pseudoalcaligenes CECT5344 in response to industrial cyanide-containing 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 pseudoalcaligenes 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 concentrations 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 analysis by LC-MS/MS has been carried out in P. pseudoalcaligenes 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-containing 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. pseudoalcaligenes CECT5344. The strain CECT5344 has been used in a batch reactor to remove at pH 9 the different 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 concentration of cyanide present in a heterogeneous mixture from an industrial source.

Isolation of bacterial strains able to degrade biphenyl, diphenyl ether and the heat transfer fluid used in thermo-solar plants.

Thermo-solar plants use eutectic mixtures of diphenyl ether (DE) and biphenyl (BP) as heat transfer fluid (HTF). Potential losses of HTF may contaminate soils and bioremediation is an attractive tool for its treatment. DE- or BP-degrading bacteria are known, but up to now bacteria able to degrade HTF mixture have not been described. Here, five bacterial strains which are able to grow with HTF or its separate components DE and BP as sole carbon sources have been isolated, either from soils exposed to HTF or from rhizospheric soils of plants growing near a thermo-solar plant. The organisms were identified by 16S rRNA gene sequencing as Achromobacter piechaudii strain BioC1, Pseudomonas plecoglossicida strain 6.1, Pseudomonas aeruginosa strains HBD1 and HBD3, and Pseudomonas oleovorans strain HBD2. Activity of 2,3-dihydroxybiphenyl dioxygenase (BphC), a key enzyme of the biphenyl upper degradation pathway, was detected in all isolates. Pseudomonas strains almost completely degraded 2000ppm HTF after 5-day culture, and even tolerated and grew in the presence of 150,000ppm HTF, being suitable candidates for in situ soil bioremediation. Degradation of both components of HTF is of particular interest since in the DE-degrader Sphingomonas sp. SS3, growth on DE or benzoate was strongly inhibited by addition of BP.

Complete genome sequence of the cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344, a Gram-negative bacterium isolated from the Guadalquir River (Córdoba, Spain), is able to utilize different cyano-derivatives. Here, the complete genome sequence of P. pseudoalcaligenes CECT5344 harboring a 4,686,340bp circular chromosome encoding 4513 genes and featuring a GC-content of 62.34% is reported. Necessarily, remaining gaps in the genome had to be closed by assembly of few long reads obtained from PacBio single molecule real-time sequencing. Here, the first complete genome sequence for the species P. pseudoalcaligenes is presented.

Draft whole genome sequence of the cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 is a Gram-negative bacterium able to tolerate cyanide and to use it as the sole nitrogen source. We report here the first draft of the whole genome sequence of a P. pseudoalcaligenes strain that assimilates cyanide. Three aspects are specially emphasized in this manuscript. First, some generalities of the genome are shown and discussed in the context of other Pseudomonadaceae genomes, including genome size, G + C content, core genome and singletons among other features. Second, the genome is analysed in the context of cyanide metabolism, describing genes probably involved in cyanide assimilation, like those encoding nitrilases, and genes related to cyanide resistance, like the cio genes encoding the cyanide insensitive oxidases. Finally, the presence of genes probably involved in other processes with a great biotechnological potential like production of bioplastics and biodegradation of pollutants also is discussed.

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an enzyme that is not essential for cyanide assimilation.

Cyanase catalyzes the decomposition of cyanate into CO(2) and ammonium, with carbamate as an unstable intermediate. The cyanase of Pseudomonas pseudoalcaligenes CECT5344 was negatively regulated by ammonium and positively regulated by cyanate, cyanide, and some cyanometallic complexes. Cyanase activity was not detected in cell extracts from cells grown with ammonium, even in the presence of cyanate. Nevertheless, a low level of cyanase activity was detected in nitrogen-starved cells. The cyn gene cluster of P. pseudoalcaligenes CECT5344 was cloned and analyzed. The cynA, cynB, and cynD genes encode an ABC-type transporter, the cynS gene codes for the cyanase, and the cynF gene encodes a novel sigma(54)-dependent transcriptional regulator which is not present in other bacterial cyn gene clusters. The CynS protein was expressed in Escherichia coli and purified by following a simple and rapid protocol. The P. pseudoalcaligenes cyanase showed an optimal pH of 8.5 degrees C and a temperature of 65 degrees C. An insertion mutation was generated in the cynS gene. The resulting mutant was unable to use cyanate as the sole nitrogen source but showed the same resistance to cyanate as the wild-type strain. These results, in conjunction with the induction pattern of the enzymatic activity, suggest that the enzyme has an assimilatory function. Although the induction of cyanase activity in cyanide-degrading cells suggests that some cyanate may be generated from cyanide, the cynS mutant was not affected in its ability to degrade cyanide, which unambiguously indicates that cyanate is not a central metabolite in cyanide assimilation.