Nitrate-responsive NarX-NarL represses arginine-mediated induction of the Pseudomonas aeruginosa arginine fermentation arcDABC operon

Abstract

Denitrification and arginine fermentation are major parts of the anaerobic metabolism of Pseudomonas aeruginosa, which is important for biofilm formation and infection. The twocomponent regulatory system NarX-NarL is part of the underlying network and is required for denitrifying growth. All target promoters identified so far are activated by NarL. In this study the effect of NarL on arginine fermentation was investigated using proteome, Northern blot and lacZ reporter gene analyses. NarL-dependent repression of the arcDABC operon was observed and the corresponding NarL-binding site in the arcD promoter region was functionally localized at "60 bp upstream of the transcriptional start site using site-directed promoter mutagenesis and reporter gene fusion experiments. The results clearly show that in the presence of nitrate NarL represses the arginine-dependent activation of the arcDABC operon mediated by ArgR. It does not influence the oxygen-tension-dependent activation via Anr. Thus, the anaerobic energy metabolism of P. aeruginosa is coordinated via NarX-NarL activity. In the presence of nitrate the highly efficient denitrification is preferred over the less attractive arginine fermentation. INTRODUCTIONIn Pseudomonas aeruginosa anaerobic metabolism is important for biofilm growth and for the infection of the cystic fibrosis lung involving biofilm-like P. aeruginosa microcolonies (Barraud et al., 2006;Filiatrault et al., 2006;Hassett et al., 2002;Palmer et al., 2007;Platt et al., 2008;Sauer et al., 2002;Van Alst et al., 2007;Worlitzsch et al., 2002;Xu et al., 1998). In the absence of oxygen P. aeruginosa grows either by denitrification or by arginine fermentation (Carlson & Ingraham, 1983;Vander Wauven et al., 1984;Zumft, 1997). The expression of genes which encode enzymes for denitrification in P. aeruginosa are controlled by the regulatory proteins Anr and Dnr, as well as the nitrate-responsive two-component system NarXNarL (Arai et al., 1997;Schreiber et al., 2007;Ye et al., 1995). Enzymes required for arginine fermentation are encoded by genes which are organized in the operon arcDABC (Luthi et al., 1990). Anaerobic expression of arcDABC has been shown to be Anr-dependent and further stimulated by the arginine-responsive regulator ArgR (Lu et al., 1999). The global oxygen-sensing regulator Anr is an Escherichia coli Fnr homologue and is essential for fermentative and denitrifying growth in the absence of oxygen (Ye et al., 1995). Moreover, Anr controls the expression of dnr, which encodes the nitrogen-oxidesensing regulator Dnr (Arai et al., 1997). Dnr finally induces expression of the genes encoding the nitrite, nitric oxide and nitrous oxide reductases required for denitrification (Arai et al., 1999(Arai et al., , 2003. Mutant and gene expression studies in P. aeruginosa and Pseudomonas stutzeri showed that the two-component system NarXNarL is additionally required for denitrifying growth (Härtig et al., 1999;Schreiber et al., 2007). The P. aeruginosa NarX protein is a sensor kinase and shares 38 % and 3...

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