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 ⌬narK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants were found to be severely restricted in nitratedependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ⌬narK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ⌬narK2::Gm and ⌬narK1K2::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. Denitrification involves four separate nitrogen oxide reductases and ultimately reduces nitrate to dinitrogen (37). Respiratory nitrate reductase, which is the first enzyme in this denitrification pathway, has its active site on the cytoplasmic side of the membrane (23). The enzyme substrate, nitrate, is an ion and cannot be taken up by the simple process of passive diffusion (18). Both of these factors require the bacterium to synthesize a transport protein(s) to carry nitrate into the cytoplasm, where the reduction of nitrate to nitrite takes place. It has been demonstrated for Pseudomonas aeruginosa, Pseudomonas stutzeri, and Escherichia coli (7,11,24) that the product of nitrate respiration, i.e., nitrite, is immediately excreted to the external environment, presumably protecting the organism from potential toxic effects. These toxic effects are due to the ability of this anion to bind to the heme groups in electron carriers, thereby inhibiting the flow of electrons (25). Genetic and physiological data suggest that nitrate transport in some bacteria occurs through two different uptake systems. Thus, for the process of nitrate assimilation, ABC transporters as well as secondary transporters are postulated to be used. On the other hand, anaerobically, for the purpose of nitrate respiration, it is postulated that bacteria rely solely on secondary transporters (18).Originally, John (14) demonstrated that membrane permeabilization of the cells significantly enhanced nitrate uptake, suggesting the need for a transport protein specific for nitrate. This was corroborated by sever...

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

“…In nitrate respiration, nitrate has to be transported into the cytoplasm, and cytotoxic nitrite has to be promptly returned to the periplasm to minimize damage to the cytoplasmic components, so integration of NarK 2 into the protein network is not unexpected (31). Interestingly, no detectable interaction of the various proteins tested with the periplasmic nitrate reductase NapAB was observed.…”

“…The final step of heme d 1 biosynthesis, localized mainly in the periplasm, involves the uroporphyrin III c-methyltransferase NirE (28), the siroheme decarboxylase NirDL/NirGH (29), and a novel electron-bifurcating dehydrogenase NirN (30). Nitrate from the environment gets transported into the cytoplasm by the nitrate/nitrite antiporter NarK2 and converted by the activity of NarGHI into nitrite, which then returns via NarK2 into the periplasm to serve as the substrate for NirS (31). Then, the integral cytoplasmic membrane protein nitric oxide reductase NorBC catalyzes the following reaction: 2NO ϩ 2H ϩ ϩ 2e Ϫ ¡ N 2 O ϩ H 2 O.…”

Influence of thePseudomonasQuinolone Signal on Denitrification inPseudomonas aeruginosa

“…During the denitrification process, NO 3 Ϫ reductase (NAR), NO 2 Ϫ reductase (NIR), NO reductase (NOR), and N 2 O reductase (NOS) are involved (61). Each denitrifying enzyme is (2,3,29,47). In order to examine the effect of PQS on transcription of each denitrifying enzyme, promoter fusion plasmids (52) with the xylE gene fused with promoter regions of narK1, nirS, norC, and nosR denitrifying genes were each transferred to a ⌬pqsA mutant.…”

Quorum Sensing Regulates Denitrification in Pseudomonas aeruginosa PAO1

“…To study whether denitrification is regulated at the transcriptional level, transcriptional fusion plasmids were constructed. During NO 3 Ϫ reduction to N 2 , NAR, NO 2 Ϫ reductase (NIR), NO reductase (NOR), and N 2 O reductase (NOS) are involved, and the genes encoding each of those enzymatic activities are organized into distinct operons (1,2,10,24). The promoter regions of narK1, nirS, norC, and nosR were fused to a xylE gene on a pMEX9 plasmid, which was constructed by replacing a lacZ␣ gene of pME4510 (22) with xylE.…”