Artificial Control of Nitrate Respiration through the lac Promoter Permits the Assessment of Oxygen-Mediated Posttranslational Regulation of the nar Operon in Pseudomonas aeruginosa

Abstract

In this study, oxygen and nitrate regulation of transcription and subsequent protein expression of the unique narK1K2GHJI respiratory operon of Pseudomonas aeruginosa were investigated. Under the control of P LAC , P. aeruginosa was able to transcribe nar and subsequently express methyl viologen-linked nitrate reductase activity under aerobic conditions without nitrate. Modulation of P LAC through the LacI repressor enabled us to assess both transcriptional and posttranslational regulation by oxygen during physiological whole-cell nitrate reduction.Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium capable of growth and/or survival anaerobically through arginine catabolism (36), pyruvate fermentation (6), or denitrification in the presence of nitrogen oxides (37). The latter process allows this organism to persist in soil as part of the global nitrogen cycle. Additionally, denitrification has been implicated in infections by this opportunistic pathogen in the airways of cystic fibrosis patients (9,18,35).During anaerobic growth of Escherichia coli, the Fnr protein is responsible for activation of the synthesis of anaerobic respiratory enzymes such as nitrate reductase (28,31). In addition, the presence of external nitrate induces the transcription of the nitrate reductase operon through the dual twocomponent regulatory systems of narX-narL (31, 32) and narQnarP (3,21,22). Parallel studies of P. aeruginosa have resulted in the characterization of a unique nar operon (24, 27) regulated by the proteins Anr and Dnr (40) as well as narX and narL (24). However, a narQ homologue has not been identified (30,34).Posttranslationally, oxygen also has the capacity to inhibit denitrification immediately at the level of nitrate uptake and nitrite excretion (10,11,33) as well as through the diversion of electron flow to oxygen in E. coli and in Paracoccus denitrificans (4, 33). Despite these studies, an experimental method for the measurement of posttranslational regulation by oxygen has been lacking.In the present study, the effects of oxygen and nitrate on the expression of the narK1K2GHJI operon (27) were examined during aerobic or anaerobic growth with and without nitrate. In addition, a P LAC element was inserted upstream of the respiratory nitrate reductase genes (narK2GHJI) of P. aeruginosa to overcome transcriptional regulation of the nar operon by oxygen and nitrate. The levels of transcription, respiratory nitrate reductase activity, and whole-cell physiological reduction of nitrate to nitrite were measured under both aerobic and anaerobic conditions, thus allowing quantitative assessment of posttranslational regulation by oxygen.The bacterial strains and plasmids used in this study are listed in Table 1. All bacteria were grown at 37°C from singlecolony isolates or overnight cultures in Luria-Bertani (LB) broth (Fisher Scientific, Pittsburgh, PA). The medium was supplemented with 1% (wt/vol) KNO 3 (LB-NO 3 ) when indicated. Aerobic cultures were set up as 50-ml volumes of LB or LB-NO 3 in a 500-ml Erlenm...