Essential role of cytochrome bd-related oxidase in cyanide resistance of Pseudomonas pseudoalcaligenes CECT5344

Abstract

Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.

FIG. 1.

Growth of P. pseudoalcaligenes CECT5344 (A) and the CIO1 mutant (B) in minimal media. Cells were precultured in 2 mM ammonium media until nitrogen was exhausted (OD₆₀₀ ≈ 0.3). Arrows indicate the addition of 1 mM of nitrogen source in the form of ammonium (•), cyanide from jewelry residue (○), or sodium cyanide (▾). One flask was kept without N addition as a control of nitrogen-free medium (▵).

FIG. 2.

Effect of cyanide, potassium, and sodium in the respiratory activity of P. pseudoalcaligenes CECT5344 membrane cell extracts. Cells were precultured in 2 mM ammonium medium until nitrogen was exhausted, and after 4 h of growth with 2 mM of cyanide from jewelry residue, the membrane fractions were obtained by ultracentrifugation. NADH oxidation activities were determined as described in Materials and Methods with the additions indicated. The bars represent the average of three independent experiments.

FIG. 3.

Induction of the cyanide-insensitive NADH oxidase activity in P. pseudoalcaligenes CECT5344 (A) and the CIO1 mutant (B). Cells were precultured in 2 mM ammonium medium until nitrogen was exhausted. The cultures were separated in four flasks, and after treatment for 4 h with 2 mM of ammonium (NH₄Cl), nitrogen free medium (−N), 2 mM of potassium cyanide (KCN), or 2 mM of cyanide from jewelry residue (RCN), the membrane fractions were obtained as described in Materials and Methods. The NADH oxidase activities were determined after addition of 2 mM KCN alkalinized with KOH (black bars) or 4 mM KCl as controls (gray bars). Prot, protein.

FIG. 4.

Map of the cio genomic region from P. pseudoalcaligenes CECT534. Genes identified in the region are represented by arrows, and the line below corresponds to the restriction map. Sl, SalI; N, NcoI; E, EcoRI; Sc, SacI; X, XhoI; P, PstI; and K, KpnI. The lower scheme shows the positions of the primers used in this work. Boxed agarose gel images contain the RT-PCR results obtained with RNA purified from ammonium-grown cells (A) or cyanide-grown cells (CN⁻).

FIG. 5.

Reduced minus oxidized difference spectra of membranes from P. pseudoalcaligenes CECT5344 (A and B) and CIO1 mutant (C and D). Cells were precultured in 2 mM ammonium medium until nitrogen was exhausted. The cultures were separated in two flasks, and after treatment for 4 h with 2 mM of ammonium (A and C) or 2 mM potassium cyanide (B and D), the membrane fractions were obtained as described in Materials and Methods. The spectra were recorded in 100 mM Tris (pH 8.0), and the protein concentration was 3 mg/ml in all cases.