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

Abstract

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.

FIG. 1.

Induction of cyanase activity in media containing both ammonium and cyanate simultaneously. Cells were cultured in 1 liter of M9 media containing ammonium and cyanate at a 5-mM final concentration as N sources. At the indicated times, the amount of cell growth (OD_{600 nm}) was determined and 50 ml of the cultures was harvested by centrifugation and used to determine the level of cyanase activity (dashed bars) as indicated in Materials and Methods. Cyanate and ammonium concentrations in the supernatants were determined. The data correspond to a single experiment, and two other independent experiments gave similar results.

FIG. 2.

Effect of cyanate concentration on the induction of cyanase activity. The cells were pregrown with 5 mM nitrate as the sole nitrogen source and were collected by centrifugation at the mid-exponential growth phase. After being washed twice in nitrogen-free media, the cells were resuspended in fresh media up to an OD₆₀₀ of 0.35. The culture was separated into six flasks that were treated with increasing amounts of cyanate. One flask, kept as a control, had no cyanate added (-N), and 5 mM nitrate was added to another flask (Nitrate). At the indicated times, 50-ml aliquots from each culture were collected, and the cyanase activities in the corresponding cell extracts were measured. The experiment was repeated three times with similar results.

FIG. 3.

The 5.7-kb cyn gene cluster of P. pseudoalcaligenes CECT5344 involved in cyanate assimilation. The 1.5-kb SalI (S) DNA fragment includes 0.7 kb of the hemE gene, a 143-bp noncoding region, the whole cynS gene, and 0.3 kb of the cynD gene. The 2.8-kb SacI (Sc) fragment comprises 0.4 kb of the hemE gene, the whole cynS, cynD, and cynB genes, and 116 bp of the cynA gene. The 4.8-kb ApaI (Ap)/SmaI (Sm) fragment includes 231 bp of the cynB gene, the whole cynB, cynA, and cynF genes, and 191 bp of the gltD gene. Between the cynA and cynF genes, a 268-bp promoter region is found. The 1.7-kb and 3.8-kb PCR fragments mentioned in the text are also shown. The position of a putative transcription terminator downstream from the cynS gene is also indicated.

FIG. 4.

(A) Multiple amino acid sequence alignment of cyanases from several bacteria, P. pseudoalcaligenes CECT5344 (EF 451798), P. putida F1 (YP_001268549), P. aeruginosa 2192 (ZP_00975588), S. elongatus PCC 6301 (YP_172699), P. marinus strain NATL1A (YP_001013899), and E. coli O157:H7 (P58704). Identical residues are in black, and similar residues are in gray. The R, E, and S residues of the catalytic triad are marked by asterisks. (B) Phylogenetic tree of bacterial cyanases. In addition to the bacterial strains described for panel A, the tree includes Burkholderia cepacia AMMD (YP_777825), P. aeruginosa PA7 (NP-250742), Pseudomonas syringae pv. phaseolicola 1448A (YP_275568), P. fluorescens Pf0-1 (YP_349098), Synechococcus sp. BL107 (ZP_01469110), Thiomicrospira crunogena (YP_390311), Synechococcus sp. WH 8102 (NP_898579), Pseudomonas stutzeri (YP_001174036), Marinobacter algicola (ZP_01892318), Roseovarius sp. 217 (ZP_01036876), Ralstonia metallidurans (YP_587992), and Reinekea sp. (ZP_01113316). The tree was generated with MEGA 4.1 software.

FIG. 5.

Tolerance of the wild type (white bars) and cynS⁻ mutant (dashed bars) strains of P. pseudoalcaligenes CECT5344 to cyanate. Cells were cultured in mineral medium with 10 mM potassium nitrate as the nitrogen source supplemented with the indicated concentration of cyanate. The optical densities of the cultures at 600 nm (OD_{600 nm})were taken 48 h after inoculation. The experiments were run in triplicate.

FIG. 6.

Purification of the cyanase from P. pseudoalcaligenes CECT5344 expressed in E. coli DH5α. Cyanase was heterologously expressed in E. coli and purified as indicated in Materials and Methods. The different lanes in the gel subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis correspond to molecular weight markers (lane 1), the cell extract (lane 2), the supernatant after centrifugation of a heated (70°C for 30 min) cell extract (lane 3), and 45% to 60% of the ammonium sulfate fraction of the heated extract (lane 4). The arrow indicates the location of the cyanase on the gel.