Identification and characterization of two genes common to the two nitrate reduction systems of Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is able to use nitrate for a nitrogen source (assimilation) and a terminal electron acceptor (anaerobic respiration). It has been demonstrated that there are genes common to both nitrate reductase systems in P. aeruginosa. One set of genes designated snr (s&barbelow;hared n&barbelow;itrate r&barbelow;eduction) have been recently cloned and partially characterized.;I our first study, we demonstrated that the snr-1 gene encodes for a predicted 52.5 kDa protein that is 82% similar to a unique cytochrome c of Desulfomonile tiedjei DCB-1. Aaerobic growth rates were significantly higher in the snr-1 mutant harboring a multicopy plasmid containing snr-1. In contrast, aerobic growth rates of the restored the mutant using nitrate as the sole nitrogen source was similar to those of wild type. This information suggests that Snr-1 plays a valuable and vital role for both forms of nitrate reduction possibly as an electron donor.;In our second study, we determined that during anaerobic respiration the expression of snr-1 was negatively controlled by the rhl component of P. aeruginosa quorum-sensing circuitry. Under anaerobic conditions a lasR rhlR and rhlR mutants exhibited a 4-fold greater snr-1::lacZ activity than the wild type. A time-course study revealed a decrease in snr-1::lacZ activity as the population increased in the wild-type strain but not in a lasR rhlR mutant. We were also able to identify a lux box approximately 100-by upstream of the snr-1 transcriptional start site. Finally, we demonstrated that the lasR rhlR and rhlR mutants grew significantly faster under anaerobic conditions when compared to the wild type.;Our last study identifies a second set of shared genes involved in both forms of nitrate reduction in P. aeruginosa. The mol genes are involved in the uptake of molybdate and the synthesis of the activated molybdenum cofactor (MoCo). We have identified a 21.8-kDa protein, which is 31% identical to the MobA protein of E. coli. An isogenic mobA::Gm mutant was unable to grow either aerobically or anaerobically in the presence of nitrate. The addition of the purified MobA protein to cell extracts devoid of nitrate reductase activity restored wild-type activity. (Abstract shortened by UMI.).