Involvement of NarK1 and NarK2 proteins in transport of nitrate and nitrite in the denitrifying bacterium Pseudomonas aeruginosa PAO1

Abstract

Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the DeltanarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the DeltanarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

FIG. 1.

Map of the narK1K2GHJI operon of Pseudomonas aeruginosa. The map shows the narK1 and narK2 genes to be upstream of the structural genes of nitrate reductase (narGHJI). Relevant restriction sites used to create deletions are shown. The endogenous promoter for the operon is shown as P_nar. The direction of transcription of both the operon and the gentamicin cassette (Gm) is shown with the help of arrows. The orientation of the Gm cassette in the gene disruptions was always positive with respect to the gene, as shown in the figure. The figure is not drawn to scale. The ΔnarK1::Gm mutant was created by blunt-ending the Gm cassette into the NcoI-SalI deletion site. The ΔnarK2::Gm mutant was created by blunt-ending the Gm cassette into the XhoI-ApaI deletion site. The ΔnarK1K2::Gm mutant was created by blunt-ending the Gm cassette into the NotI-ApaI deletion site.

FIG. 2.

Anaerobic growth of Pseudomonas aeruginosa PAO1 in LB medium supplemented with nitrate. All the inocula were prepared by growing the strains overnight in shaker-grown starter cultures in LB medium, which were then transferred to LB medium supplemented with 1% nitrate and the appropriate concentrations of gentamicin and/or carbenicillin and switched to anaerobic conditions using oxyrase and argon gas. (A) Anaerobic growth of PAO1 (⋄), ΔnarK1::Gm strain (▪), and ΔnarK1::Gm/pnarK1 complemented strain (narK1 complement) (▴). (B) Anaerobic growth of PAO1 (⋄), ΔnarK2::Gm strain (▪), and ΔnarK2::Gm/pnarK2 complemented strain (narK2 complement) (▴). (C) Anaerobic growth of PAO1 (⋄), ΔnarK1K2::Gm strain (▪), and ΔnarK1K2::Gm/pnarK1K2 complemented strain (narK1K2 complement) (▴).

FIG. 3.

Complementation of the ΔnarK2::Gm mutant and the ΔnarK1K2::Gm mutant with pnarK. The narK gene cloned into the pUCP18 plasmid vector was obtained from E. coli K-12. All strains were grown overnight in LB medium and were then transferred to LB medium supplemented with 1% nitrate and an appropriate concentration of gentamicin and carbenicillin and switched to anaerobic conditions. The anaerobic growth of PAO1 (⋄), ΔnarK2::Gm strain complemented with pnarK (▪), and ΔnarK1K2::Gm strain complemented with pnarK (▴) is shown.