Expanding And Refining A Model For Denitrification In Rhodobacter Sphaeroides 2.4.3
Rhodobacter sphaeroides strain 2.4.3 is a photoheterotroph of the αproteobacteria. It is metabolically versatile and in addition to anoxygenic photosynthesis and aerobic respiration, it is capable of denitrification. The denitrification system of strain 2.4.3 has been studied in detail by our lab. Here, we expand on our model for denitrification. The genome sequence of strain 2.4.3 revealed the presence of two periplasmic nitrate reductases. Using expression and mutant studies, we show that one of these enzymes is expressed aerobically and is involved in aerobic redox homeostasis. The second enzyme is expressed under low oxygen conditions and is the nitrate reductase involved in denitrification. We provide evidence for oxygen sensitivity of the denitrification regulator NnrR in a mutant lacking the high oxygen affinity cytochrome oxidase. NnrR is a member of the FNR/CFP family of transcriptional regulators that has been shown to regulate denitrification genes in response to the denitrification intermediate nitric oxide. Using the predicted DNA binding motif of NnrR we analyzed the genome for genes potentially under NnrR control. We identified five new genes and demonstrate that expression is dependent on NnrR. One of these genes encodes pseudoazurin, which is shown to be an electron donor to nitrite reductase along with a cytochrome. Two other genes identified encode NorEF, putative additional subunits of the nitric oxide reductase enzyme complex. Inactivation of these genes decreases denitrification activity and cells show greater sensitivity to nitric oxide. We also investigated denitrification-independent nitric oxide signaling and metabolism. The Sphaeroides Heme Protein (SHP) has been shown to have nitric oxide dioxygenase activity. Here, we show that shp is induced under stationary phase photo-growth and that the shp mutant is not impaired in growth under these conditions. Finally, we attempt to characterize a nitric oxide binding protein that may mediate signaling through a diguanylate cyclase. This may link nitric oxide into the cellular cyclic-diguanosine monophosphate signaling pathway.
dissertation or thesis