UC Riverside

Microorganisms capable of nitrifier denitrification and aerobic denitrification are of significant interest due to their release of NO and N₂O in the atmosphere. Studies have therefore focused on the physiology and genetic diversity in organisms capable of nitrogen oxide (NOx) metabolism in the production of these gases. Genomic analysis of ammonia-oxidizing bacteria (AOB) revealed the presence of NOx-related gene homologues normally present in denitrifying bacteria. Thought to serve in NO detoxification, these genes include nitrite reductase (nirK), nitric oxide reductases (norB and norS), and cytochromes P460 (cytL) and c' beta (cytS). The goal of the study in Chapter 2 was to determine regulation of these genes in three representative AOB grown in HEPES- or phosphate- buffered media in the presence of 0, 10, and 20mM NaNO₂. Results indicate that differential regulation of homologous genes occurs in each AOB in response to nitrite, suggesting different mechanisms in coping with nitrosative stress.

Sphingomonas wittichii RW1 contains genes involved in NOx metabolism, including nirK, norB, norZ, and hmp (encoding flavohemoglobin), but has no previous history of denitrification. The goal of the study in Chapter 3 was to determine whether these genes were regulated in response to nitrite under initial 20% and 3% oxygen levels. S. wittichii RW1 was not capable of nitrite respiration. High basal mRNA levels of these genes indicate differential regulation by oxygen. Increased levels of hmp and norB mRNA in NO₂^- -treated groups under low O₂ suggest hypoxic function and activity. Total nitrite consumption was observed, and aerobic production of N₂O was greatest in treated cells incubated under 3% O₂. Thus, S. wittichii is capable of aerobic denitrification, in the detoxification of nitrite.

A Delta;norZ mutant was constructed in S. wittichii for the study in Chapter 4 to determine the roles of the two nor genes with respect to aerobic N₂O production. Total nitrite consumption occurred indicating a functional activity of nirK. The Delta;norZ strain was able to produce N₂O, though significantly less compared to wildtype cells. An increase in norB mRNA levels in NO₂^- -treated cells was observed, suggesting compensation for the loss of NorZ function.

The studies presented show that nitrite differentially affected transcription of nirK, amoA, and norS in the AOB. In S. wittichii, NOx- related genes are regulated by oxygen and collectively used to aerobically detoxify nitrite, resulting in N₂O production; additionally, norB is an isofunctional NO reductase.