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Journal of Bacteriology, September 2007, p. 6501-6505, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00491-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Chris E. Noriega,{dagger} Vandana Sharma,{ddagger} and John J. Rowe*

Department of Biology, University of Dayton, Dayton, Ohio 45469

Received 30 March 2007/ Accepted 22 June 2007


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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 PLAC, P. aeruginosa was able to transcribe nar and subsequently express methyl viologen-linked nitrate reductase activity under aerobic conditions without nitrate. Modulation of PLAC through the LacI repressor enabled us to assess both transcriptional and posttranslational regulation by oxygen during physiological whole-cell nitrate reduction.


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TEXT
 
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 two-component regulatory systems of narX-narL (31, 32) and narQ-narP (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 PLAC 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 single-colony isolates or overnight cultures in Luria-Bertani (LB) broth (Fisher Scientific, Pittsburgh, PA). The medium was supplemented with 1% (wt/vol) KNO3 (LB-NO3) when indicated. Aerobic cultures were set up as 50-ml volumes of LB or LB-NO3 in a 500-ml Erlenmeyer flask by inoculating 500 µl of cells and shaking at 250 rpm to an optical density at 600 nm of 0.3 to 0.4. Cultures used to measure anaerobic transcription of narG and to conduct in vitro anaerobic respiratory nitrate reductase assays were first grown aerobically to an optical density at 600 nm of 0.3 to 0.4, after which time they were shifted to complete anaerobic conditions (BBL Anaerobic GasPak System) for 3 h. For anaerobic growth cultures and nitrite excretion studies, 1 ml of mid-log-phase cells was inoculated in 100-ml volumes of LB-NO3 and incubated with magnetic stirring in a 125-ml Erlenmeyer flask with rubber stoppers equipped with ports for sample withdrawal and one-way gas release valves. To ensure complete anaerobiosis of the system, the medium was supplemented with 2% (wt/vol) Oxyrase (Oxyrase, Inc., Mansfield, OH) and flushed with argon. The antibiotics (BioWorld, Dublin, OH) used for E. coli were as follows: ampicillin (100 µg/ml), gentamicin (15 µg/ml), and tetracycline (25 µg/ml). Gentamicin (300 µg/ml) and carbenicillin (500 µg/ml) were used for P. aeruginosa.


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  		TABLE 1. Strains, plasmids, and oligonucleotide primers

For the construction of strain JVC, a 1.5-kb SmaI digest containing Gmxlac was blunt ended into the NotI site of pEX18Ap::narK1K2. Single-copy, chromosomal gene disruptions were created using a gene replacement technique previously described (25, 29). loxP excision of the gentamicin resistance marker was conducted by transformation with pCRE into a P. aeruginosa strain with a narK1-Gmxlac disruption. Several passages of growth in LB containing 500 µg/ml carbenicillin were followed by selection for the loss of growth in LB containing 300 µg/ml gentamicin. The pCRE plasmid was cured from the strain through one passage in LB, followed by growth in LB containing 5% sucrose and selection for carbenicillin sensitivity. Constructs were confirmed by PCR using primers listed in Table 1.

JVC was constructed by inserting the Gmxlac cassette within narK1 for two reasons. First, it has been shown for P. aeruginosa that while narK1K2GHJI is transcribed as an operon, only NarK2 is required for respiratory nitrate reduction (27). Second, we did not want to interfere with physiologically regulated anaerobic transcription of the nar promoter since expression levels manipulated through a lac promoter could be either limiting or overexpressed and, thus, will not reflect physiological responses. Using the same reasoning, transcriptional termination sequences were not placed upstream of PLAC in the pUCGmxlac vector. Thus, the transcriptional activity of narK2GHJI would be under the control of both the nar promoter and the PLAC element during anaerobic conditions and be under the control of solely PLAC during aerobic conditions.

Transcription of nar under aerobic and anaerobic conditions in JVC (narK1-PLAC). To test the ability of PLAC to aerobically transcribe narK2GHJI, the mRNA levels of narG in PAO1 and JVC were measured through quantitative reverse transcription-PCR in LB with or without nitrate in aerobic and anaerobic conditions. The results further corroborated the previous experimental proof that in P. aeruginosa, narK1 and narK2 were contained in the nar operon (27) and demonstrated that the presence of nitrate alone was not sufficient for the transcription of narG in wild-type PAO1 and that anaerobic conditions were required (Table 2). In contrast, JVC transcribed narG aerobically in both the presence and the absence of nitrate (15.5 ± 3.8 and 23.4 ± 6.0 relative severalfold increases, respectively). The level of narG transcription anaerobically was significantly higher for both PAO1 and JVC since the operon was under the physiological control of the nar promoter. Finally, we manipulated the transcription of narG by transforming a plasmid containing lacIq (pREP) into JVC. As expected, the addition of pREP significantly reduced aerobic narG transcriptional levels in JVC (Table 2).


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  		TABLE 2. Transcriptional levels of narG in P. aeruginosa PAO1 and JVC

The addition of PLAC upstream of narK2GHJI enabled us to constitutively transcribe the operon in both the presence and the absence of oxygen and nitrate and to modulate aerobic transcriptional activity by utilizing pREP.

Respiratory nitrate reductase activity. To determine whether the same trends are observed at the enzyme level, methyl viologen-linked respiratory nitrate reductase activity was measured (14) in cell extracts of strains grown in LB with or without nitrate aerobically and anaerobically (Table 3). The results indicated that enzyme activity was detected only in wild-type PAO1 under anaerobic conditions in the presence of nitrate (Table 3), confirming the narG transcriptional activities measured in Table 2 and the requirement of both nitrate and anaerobiosis for expression of the respiratory nitrate reductase. P. aeruginosa also contains a periplasmic nitrate reductase (38), but we did not detect any enzyme activity for P. aeruginosa under aerobic conditions (17, 27). Nitrate reductase activity in JVC was consistently detected even in the absence of nitrate and under aerobic conditions due to the expression of narGHJI through PLAC (Table 3). Eighty percent of the specific activity detected aerobically without nitrate was retained within the insoluble fraction of the crude extract, indicating that the aerobic nitrate reductase measured was predominantly membrane bound (data not shown). Anaerobically, the nitrate reductase activity of JVC was approximately twice the level observed in wild-type PAO1 (1,120 ± 150 and 560 ± 77 nmol nitrite min–1 mg–1 protein, respectively [Table 3]). We also determined the ability of pREP to inhibit nitrate reductase activity and found both aerobic and anaerobic nitrate reductase activities to be significantly reduced in JVC with pREP (Table 3). Although pREP did not completely diminish anaerobic nitrate reductase activities, the level of inhibition was sufficient to significantly hinder anaerobic growth (data not shown).


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  		TABLE 3. Nitrate reductase activities of P. aeruginosa PAO1 and JVC

Posttranslational oxygen regulation of nitrate reduction. Through quantitative reverse transcription-PCR and methyl viologen-linked respiratory nitrate reductase activity, we have shown that oxygen inhibition of transcription and translation of the nar operon was overcome by modulating transcription of narK2GHJI through PLAC. The use of pREP also allowed us to express nitrate reductase activity anaerobically in JVC over a wide range from 218 ± 7 to 1,120 ± 150 nmol nitrite min–1 mg–1 protein. More importantly, the levels of enzyme activity in JVC with pREP under anaerobic conditions are similar to those observed aerobically in JVC alone (218 ± 7 and 210 ± 9 nmol nitrite min–1 mg–1 protein, respectively). These expression levels allowed us to measure whole-cell nitrate reduction to nitrite aerobically and anaerobically in strains with comparable nitrate reductase activities and thus explore posttranslational regulation by oxygen (Table 3). We examined JVC for physiological whole-cell nitrate reduction (non-methyl viologen driven) to nitrite indirectly by measuring the levels of nitrite in the growth medium. Specifically, the supernatant was collected by centrifugation (10,000 x g, 4°C, 10 min) and measured for nitrite (16). The disappearance of nitrite was not considered since previous studies have shown that the presence of nitrate inhibits the reduction of nitrite and that its subsequent secretion by whole cells is stoichiometrically correlated to the disappearance of nitrate (5, 13). The JVC strain excreted nitrite when grown aerobically (1.5 ± 1 nmol nitrite µg–1 protein [Table 4]) while the levels of aerobic nitrite excretion in wild-type PAO1 remained undetectable. During anaerobic growth, the JVC strain containing pREP excreted nitrite at a rate approximately sixfold higher than the levels observed under aerobic conditions in the same strain without pREP (9.4 ± 0.5 and 1.5 ± 0.1 nmol nitrite µg–1 protein, respectively [Table 4]). Although similar nitrate reductase activities were observed in aerobic JVC and anaerobic JVC with pREP (Table 3), the levels of nitrite excretion were significantly lower under aerobic conditions. The PLAC insertion enabled JVC to aerobically transcribe nar and produce functional wild-type levels of respiratory nitrate reductase activity under aerobic conditions, thus clearly demonstrating the physiological significance of posttranslational regulation by oxygen at the level of nitrate transport and/or diversion of electron flow (4, 10, 11).


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  		TABLE 4. Nitrite excretion levels of P. aeruginosa PAO1 and JVC

The results in this investigation are consistent with those of past studies of transcriptional regulation of nar (1, 2, 8, 27, 39-41) that were recently challenged by a DNA microarray-based study (7) which reported no significant changes in narG transcription aerobically versus anaerobically in P. aeruginosa grown on complex medium supplemented with nitrate. The authors of that recent study concluded that nitrate alone was sufficient for nar expression in both aerobic and anaerobic environments.

Finally, the modification of pUCGMlox allowed us to modulate the expression of the narK2GHJI operon for physiological studies which would otherwise be difficult to perform by utilizing plasmid expression vectors currently available for P. aeruginosa. With the combination of pREP and PLAC, we were able to conditionally express nitrate reductase activities chromosomally. Intermediate levels of nitrate reductase activities can even be obtained using IPTG (isopropyl-ß-D-thiogalactopyranoside) (data not shown). Therefore, the use of this technique enables the investigator to integrate a regulatory element(s) and modulate gene expression of operons and multiple open reading frames to further take advantage of the available P. aeruginosa genome database (34).


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ACKNOWLEDGMENTS
 
We are especially grateful to Benoit Polack and Lauriane Quenee for kindly providing pUCGMlox.

This work was supported in part by the University of Dayton Summer Fellowship Program and the Department of Biology.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, University of Dayton, 300 College Park, Dayton, OH 45469-2320. Phone: (937) 229-2521. Fax: (937) 229-2021. E-mail: John.Rowe{at}notes.udayton.edu Back

{triangledown} Published ahead of print on 6 July 2007. Back

{dagger} Present address: Section of Microbiology, University of California, Davis, CA 95616-8665. Back

{ddagger} Present address: Department of Biomedical Engineering, University of California, Davis, CA 95616-5294. Back


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Journal of Bacteriology, September 2007, p. 6501-6505, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00491-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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