The correlation between nitrite reduction and nitrosation by gastric isolates of N. subflava
The work in this thesis covers the development of assays for bacterial nitrosation and nitrous oxide reductase. The use of these two assays and the nitrite reductase assay allowed a detailed study not only of the correlation between nitrite reductase and nitrosation but also between nitrous oxide reductase and nitrosation in Neisseria subflava. The three activities have been studied with various electron donors and inhibitors and also with cells grown under various conditions. Maximum activities were:- Nitrite reductase 1130 nmol/min/mg protein with lactate as electron donor, Nitrous oxide reductase 7.17 ^unol/min/mg protein with glucose as electron donor 7 and nitrosation 427 nmol/min/mg protein with ethanol as electron donor. When this work was coupled with the production of N.subflava mutants deficient in different aspects of denitrification using insertional transposon mutagenesis it provided a wealth of information regarding the correlation between denitrification and bacterial nitrosation. The strains of N.subflava isolated from the achlorhydric stomach were all able to nitrosate. The ability to nitrosate required the reduction of nitrite but nitrite reductase itself was unable to catalyse nitrosation. Reactions involving nitrous oxide, nitrous oxide reductase and dinitrogen were shown to have no involvement in bacterial nitrosation. Leaving only nitric oxide and nitric oxide reductase as possible connections between bacterial nitrosation and denitrification. With the knowledge that oxygen was required for bacterial nitrosation and the growth conditions necessary to induce bacterial nitrosation a hypothesis could be proposed to connect nitrosation activity and denitrification: Nitric oxide may accumulate following nitrite reduction and could be subsequently oxidised (by molecular oxygen) to produce dinitrogen trioxide and dinitrogen tetroxide. These two products are known to be very powerful nitrosating agents which can then react with amines, amides and ureas to produce various N-nitroso compounds. As the strains of N.subflava used in this work were gastric isolates it is plausible that these reactions producing N-nitroso compounds could occur in the stomach. This would account for the high risk from gastric cancer seen in patients whose gastric pH is close to 7 allowing bacteria to colonise the stomach.