A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344

Pseudomonas pseudoalcaligenes CECT 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 coli enabled the recombinant strain to fully assimilate 3-cyanoalanine.P. pseudoalcaligenes CECT 5344 showed a weak growth level with 3-cyanoalanine as the N source, unless KCN was also added. Moreover, anit4 knockout mutant ofP. pseudoalcaligenes CECT 5344 became severely impaired in its ability to grow with 3-cyanoalanine and cyanide as nitrogen sources. The native enzyme expressed inE. coli was 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. Thenit4 gene is located downstream of the cyanide resistance transcriptional unit containingcio1 genes, whose expression levels are under the positive control of cyanide. Real-time PCR experiments revealed thatnit4 expression was also positively regulated by cyanide in both minimal and LB media. These results suggest that this gene cluster includingcio1 andnit4 could be involved both in cyanide resistance and in its assimilation byP. pseudoalcaligenes CECT 5344.IMPORTANCE Cyanide 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. pseudoalcaligenes CECT 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. pseudoalcaligenes . This 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.