title: A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344.
abstract: Pseudomonas pseudoalcaligenesCECT 5344 is a bacterium able to assimilate cyanide as a sole nitrogen source. Under this growth condition, a 3-cyanoalanine nitrilase enzymatic activity was induced. This activity was encoded bynit4, one of the four nitrilase genes detected in the genome of this bacterium, and its expression inEscherichia colienabled the recombinant strain to fully assimilate 3-cyanoalanine.P. pseudoalcaligenesCECT 5344 showed a weak growth level with 3-cyanoalanine as the N source, unless KCN was also added. Moreover, anit4knockout mutant ofP. pseudoalcaligenesCECT 5344 became severely impaired in its ability to grow with 3-cyanoalanine and cyanide as nitrogen sources. The native enzyme expressed inE. coliwas purified up to electrophoretic homogeneity and biochemically characterized. Nit4 seems to be specific for 3-cyanoalanine, and the amount of ammonium derived from the enzymatic activity doubled in the presence of exogenously added asparaginase activity, which demonstrated that the Nit4 enzyme had both 3-cyanoalanine nitrilase and hydratase activities. Thenit4gene is located downstream of the cyanide resistance transcriptional unit containingcio1genes, whose expression levels are under the positive control of cyanide. Real-time PCR experiments revealed thatnit4expression was also positively regulated by cyanide in both minimal and LB media. These results suggest that this gene cluster includingcio1andnit4could be involved both in cyanide resistance and in its assimilation byP. pseudoalcaligenesCECT 5344.IMPORTANCECyanide is a highly toxic molecule present in some industrial wastes due to its application in several manufacturing processes, such as gold mining and the electroplating industry. The biodegradation of cyanide from contaminated wastes could be an attractive alternative to physicochemical treatment.P. pseudoalcaligenesCECT 5344 is a bacterial strain able to assimilate cyanide under alkaline conditions, thus avoiding its volatilization as HCN. This paper describes and characterizes an enzyme (Nit4) induced by cyanide that is probably involved in cyanide assimilation. The biochemical characterization of Nit4 provides a segment for building a cyanide assimilation pathway inP. pseudoalcaligenesThis information could be useful for understanding, and hopefully improving, the mechanisms involved in bacterial cyanide biodegradation and its application in the treatment of cyanide-containing wastes.
authors.affiliation: Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.
authors.forename: Felipe
authors.initials: F
authors.lastname: Acera
authors.affiliation: Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Carmona
authors.affiliation: Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Castillo
authors.affiliation: Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.|INBio G+C Research Institute, Universidad de Extremadura, Cáceres, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Quesada
authors.affiliation: Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain rblasco@unex.es.|Meat and Meat Products Research Institute (IProCar), Universidad de Extremadura, Cáceres, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Blasco
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000619:Aminohydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D064751:Ammonium Compounds / Q000378:metabolism; D003001:Cloning, Molecular; D003486:Cyanides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D044205:Pseudomonas pseudoalcaligenes / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015533:Transcriptional Activation
chemical_list: D064751:Ammonium Compounds; D003486:Cyanides; D011994:Recombinant Proteins; C004631:3-cyanoalanine; D000619:Aminohydrolases; C021867:nitrilase; D006836:Hydro-Lyases; C019954:3-cyanoalanine hydratase; D009584:Nitrogen; D000409:Alanine
keywords: biodegradation; cyanide; nitrilase