Correlation between the results of cultures and the molecular BIOFIRE® joint infection panel in a cohort of pediatric patients with bone and joint infections in Bogotá, Colombia.

Introduction: Bone and Joint Infections (BJI) have high morbidity. Methicillin resistant Staphylococcus aureus (MRSA) has increased. Culture-based diagnosis has difficult to recovering fastidious bacteria and detecting polymicrobial infections, molecular methods offer a promising improvement for the diagnosis of BJI with reduced time to result. The aim of the study was to determine the correlation between culture results and the Biofire joint infection panel (BJIP) in a cohort of pediatric patients with BJI. Materials and methods: Descriptive study. Patients admitted with probable o confirmed BJI between July 1, 2019 and February 28, 2021 at HOMI. Blood cultures, synovial and bone fluid samples were taken. Samples were kept at -70 °C. On September 2022, the panel was performed. Results: 32 patients were included. The average age was 83m (RIQ: 32-145). 23 (71.8%) patients had a positive culture. The most frequent microorganism were S. aureus 19 (83%), 11/19 (57.9%) Staphylococci isolates were MRSA. 24/32 (75%) were positive by panel, 20 positive detections were concordant with culture, there were 6 additional isolates by panel (2 S. aureus, 2 S. pyogenes, 1 K. kingae and 1 C. albicans), three microorganisms were isolated in culture but not in the panel. (2 S. aureus and 1 S. agalactiae). Two patients with coinfection were detected. All MRSA were detected by culture and panel. In 26 (81.3%) patients the etiology was documented by any method. Conclusion: These results showed a moderate level of agreement between BJIP and culture (κ = 0.47). The panel allowed the detection of fastidious bacteria including K. kingae and polymicrobial samples. There was a very good level of agreement between the panel and culture for the MRSA detection (κ = 1).

Genomic Insights into Cyanide Biodegradation in the Pseudomonas Genus.

Molecular studies about cyanide biodegradation have been mainly focused on the hydrolytic pathways catalyzed by the cyanide dihydratase CynD or the nitrilase NitC. In some Pseudomonas strains, the assimilation of cyanide has been linked to NitC, such as the cyanotrophic model strain Pseudomonas pseudoalcaligenes CECT 5344, which has been recently reclassified as Pseudomonas oleovorans CECT 5344. In this work, a phylogenomic approach established a more precise taxonomic position of the strain CECT 5344 within the species P. oleovorans. Furthermore, a pan-genomic analysis of P. oleovorans and other species with cyanotrophic strains, such as P. fluorescens and P. monteilii, allowed for the comparison and identification of the cioAB and mqoAB genes involved in cyanide resistance, and the nitC and cynS genes required for the assimilation of cyanide or cyanate, respectively. While cyanide resistance genes presented a high frequency among the analyzed genomes, genes responsible for cyanide or cyanate assimilation were identified in a considerably lower proportion. According to the results obtained in this work, an in silico approach based on a comparative genomic approach can be considered as an agile strategy for the bioprospection of putative cyanotrophic bacteria and for the identification of new genes putatively involved in cyanide biodegradation.

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.

Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation.

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT-/- mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor ß1 (TGFß1) and collagen (COL1A1) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

Changes in the incidence of acute bacterial meningitis caused by Streptococcus pneumoniae and the implications of serotype replacement in children in Colombia after mass vaccination with PCV10.

Introduction: Acute bacterial meningitis (ABM) is a public health problem. The disease has reemerged after the introduction of pneumococcal conjugate vaccines (PCVs) due to an increase in serotypes that are not covered. The objective was to determine the changes in the disease incidence before and after the introduction of the 10-valent vaccine (PCV10) in Colombia. Methods: This multicenter study was conducted in 17 hospitals in Colombia. Data were collected from January 2008 to December 2019 in 10 hospitals in Bogotá and from January 2017 to December 2019 in seven hospitals in Cali, Medellín and Cartagena. The data were grouped into three periods: 2008-2011, 2012-2015, and 2016-2019. Results: Of the 706 cases of invasive pneumococcal disease, 81 (11.4%) corresponded to meningitis. The relative incidence in Bogotá in the first period was 0.6 per 100,000 patients ≤ 5 years, decreased to 0.4 per 100,000 patients ≤ 5 years in the second period and increased in the third period to 0.7 per 100,000 patients ≤ 5 years. Serotypes covered by PCV10 decreased from 75 to 9.1%, with Spn19A (31.8%) and Spn34 (13.6%) emerging in the third period. Increased resistance to penicillin (13 to 37%) and to ceftriaxone (5.9 to 16%) was due to the emergence of multidrug-resistant Spn19A. The total mortality rate was 23.5% and increased from 12 to 33%. Conclusions: ABM due to pneumococcus has high morbidity and mortality rates. Reemergence of the disease has been observed due to the inclusion of polymerase chain reaction (PCR) for diagnosis and replacement of circulating serotypes after the introduction of PCV10, with an increase in Spn19A, which causes death and exhibits antimicrobial resistance. Continued surveillance is needed.

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.

Association of low serum albumin with venous thrombosis in pediatric patients.

OBJECTIVES: The incidence of venous thromboembolism (VTE) in children is increasing, attributed in part to increased utilization of central venous catheters (CVCs). Children with protein losing disorders (PLDs) and low serum albumin may have an increased incidence of thrombosis. We sought to determine the prevalence of PLDs and hypoalbuminemia at the time of diagnosis of VTE in pediatric patients and its relationship to central venous catheters. METHODS: We performed a single institution retrospective study of 65 consecutive hospitalized pediatric patients with an acute VTE. Data collected included clinical diagnoses, type of thrombosis, presence or absence of a CVC, and serum albumin level, if available. RESULTS: Of 65 patients with acute VTE, 51 % (33/65) had catheter-related thrombosis (CRT), including 71 % (19/27) of patients <12 years of age and 37 % (14/38) of patients aged 12 to 23 (P = 0.008). Eleven VTEs occurred in patients with a diagnosis of a PLD; of these, ten (91 %) were CRT and one (9 %) was a non-CRT (P = 0.003). Serum albumin levels obtained within four days of diagnosis of VTE were available for 38 patients. An albumin level below the lower limit of the age-adjusted normal reference range was documented in 27/38 (71 %) patients with VTE compared to 1011/3028 (33 %) of all pediatric patients admitted to the hospital during a two-year period (P < 0.0001). Albumin levels were low in 19/22 (86 %) patients with CRT compared with 8/16 (50 %) patients with non-CRT (P = 0.019). CONCLUSION: Low serum albumin levels are highly prevalent among pediatric patients with VTE, especially in those patients with CRT.

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.

Effect of pH on the denitrification proteome of the soil bacterium Paracoccus denitrificans PD1222.

Denitrification is a respiratory process by which nitrate is reduced to dinitrogen. Incomplete denitrification results in the emission of the greenhouse gas nitrous oxide and this is potentiated in acidic soils, which display reduced denitrification rates and high N2O/N2 ratios compared to alkaline soils. In this work, impact of pH on the proteome of the soil denitrifying bacterium Paracoccus denitrificans PD1222 was analysed with nitrate as sole energy and nitrogen source under anaerobic conditions at pH ranging from 6.5 to 7.5. Quantitative proteomic analysis revealed that the highest difference in protein representation was observed when the proteome at pH 6.5 was compared to the reference proteome at pH 7.2. However, this difference in the extracellular pH was not enough to produce modification of intracellular pH, which was maintained at 6.5 ± 0.1. The biosynthetic pathways of several cofactors relevant for denitrification and nitrogen assimilation like cobalamin, riboflavin, molybdopterin and nicotinamide were negatively affected at pH 6.5. In addition, peptide representation of reductases involved in nitrate assimilation and denitrification were reduced at pH 6.5. Data highlight the strong negative impact of pH on NosZ synthesis and intracellular copper content, thus impairing active NosZ assembly and, in turn, leading to elevated nitrous oxide emissions.

Emergence of Streptococcus pneumoniae serotype 19A (Spn19A) in the pediatric population in Bogotá, Colombia as the main cause of invasive pneumococcal disease after the introduction of PCV10.

Introduction: With the use of pneumococcal conjugate vaccines(PCV), the behavior of invasive pneumococcal disease(IPD) has changed relative to serotype distribution. The introduction of these vaccines in national immunization programs has reduced the incidence of IPD, with a marked decrease in the circulation of the serotypes included in the vaccine used in each country. However, the subsequent emergence of other serotypes not included in the vaccine, such 19A in case of PCV7 and PCV10, has been documented. Materials and methods: This was case series study (2008-2017) in pediatric patients admitted to 10 hospitals in Bogota who were diagnosed with IPD. It was conducted during the transitional period of implementing the PCV10 vaccine in Colombia in 2012. Cases of bacteremic pneumococcal pneumonia, meningitis, primary bacteremia and osteoarticular infection were included. A descriptive analysis of the demographic, clinical and laboratory variables of patients with IPD by Spn19A, its trend over time, profiles of antimicrobial susceptibility and clinical outcomes was performed. Results: There were 463 cases of IPD, 315(68%) with known serotypes. The prevalence of IPD by Spn19A was 17.7%(56 cases), tending to increase over time. During 2008-2011, the prevalence was 4.4%, and during 2014-2017, it was 32.4%, The most frequent diagnosis was pneumonia(80.4%). In nonmeningeal isolates, 39.6% were not susceptible to penicillin. An increase in the resistance was observed over time. Conclusion: Spn19A is a prevalent cause of IPD in the pediatric population of the analyzed cohort, with an increasing trend of this serotype during the surveillance period after the introduction of PCV10, being the most common serotype identified in recent years.

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.

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.

Putative small RNAs controlling detoxification of industrial cyanide-containing wastewaters by Pseudomonas pseudoalcaligenes CECT5344.

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 uses free cyanide and several metal-cyanide complexes as the sole nitrogen source and tolerates high concentrations of metals like copper, zinc and iron, which are present in the jewelry wastewaters. To understand deeply the regulatory mechanisms involved in the transcriptional regulation of cyanide-containing wastewaters detoxification by P. pseudoalcaligenes CECT5344, RNA-Seq has been performed from cells cultured with a cyanide-containing jewelry wastewater, sodium cyanide or ammonium chloride as the sole nitrogen source. Small RNAs (sRNAs) that may have potential regulatory functions under cyanotrophic conditions were identified. In total 20 sRNAs were identified to be differentially expressed when compared the jewelry residue versus ammonium as nitrogen source, 16 of which could be amplified successfully by RT-PCR. As predicted targets of these 16 sRNAs were several components of the nit1C gene cluster encoding the nitrilase NitC essential for cyanide assimilation, the cioAB gene cluster that codes for the cyanide-insensitive cytochrome bd-type terminal oxidase, the medium length-polyhydroxyalkanoates (ml-PHAs) gene cluster, and gene clusters related with a global nitrogen limitation response like those coding for glutamine synthase and urease. Other targets were non-clustered genes (or their products) involved in metal resistance and iron acquisition, such as metal extrusion systems and the ferric uptake regulatory (Fur) protein, and a GntR-like regulatory family member probably involved in the regulation of the cyanide assimilation process in the strain CECT5344. Induction of genes targeted by sRNAs in the jewelry residue was demonstrated by qRT-PCR.

Exploring the Denitrification Proteome of Paracoccus denitrificans PD1222.

Denitrification is a respiratory process that produces nitrous oxide as an intermediate, which may escape to the atmosphere before its reduction to dinitrogen through the nitrous oxide reductase NosZ. In this work, the denitrification process carried out by Paracoccus denitrificans PD1222 has been explored through a quantitative proteomic analysis. Under anaerobic conditions, with nitrate as sole nitrogen source, the synthesis of all the enzymes involved in denitrification, the respiratory nitrate, nitrite, nitric oxide, and nitrous oxide reductases, was increased. However, the periplasmic and assimilatory nitrate reductases decreased. Synthesis of transporters for alcohols, D-methionine, sulfate and copper, most of the enzymes involved in the tricarboxylic acid cycle, and proteins involved in other metabolic processes like lysine catabolism, fatty acids degradation and acetyl-CoA synthesis, was increased during denitrification in P. denitrificans PD1222. As consequence, an enhanced production of the central metabolite acetyl-CoA was observed. After establishing the key features of the denitrification proteome, its changes by the influence of a competitive electron acceptor, oxygen, or competitive nitrogen source, ammonium, were evaluated.

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.