Graduate Thesis Or Dissertation
 

Transcriptome profiling of Nitrosomonas europaea grown singly and in co-culture with Nitrobacter winogradskyi

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  • In most environments, ammonia (NH₃) is oxidized to nitrate (NO₃⁻) via the intermediate nitrite (NO₂⁻). This is a microbe-driven process involving phylogenetically distinct types of microorganisms, namely, ammonia-oxidizing bacteria (AOB) and Thaumarchaea (AOA), and nitrite-oxidizing bacteria (NOB). Nitrosomonas europaea and Nitrobacter winogradskyi are the best studied AOB and NOB, respectively, with a foundation of more than 50 years of physiological and biochemical studies. Their genomes have been sequenced and annotated. There have been few studies of the two organisms grown together. The significance of this gap in knowledge lies in the fact that during nitrification in natural environments, NO₂⁻ rarely accumulates, which implies that AOB and NOB activities are intimately coordinated. However, there are alternate abiological and biological sinks for NO₂⁻, which can result in NO₃⁻ not being the end product of nitrification. In my study, I established chemostats of N. europaea and N. winogradskyi both singly and in co-culture under the same conditions (except NOB growth medium contained NO₂⁻ instead of NH₃). At steady state, estimates were made of cell densities, chemical analysis of the culture medium, and cell samples were collected for RNA extraction. Transcriptomic profiles of the co-culture and single culture of N. europaea were compared. Co-cultures had cell densities one and a half times greater (0.25-0.27 OD₆₀₀) than the sum of single chemostat cultures (0.12 OD₆₀₀ for N. europaea and 0.05 OD₆₀₀ for N. winogradskyi). Substrate-dependent oxygen uptake rates indicated that the population density of N. europaea was about 4.7-fold greater than N. winogradskyi in the co-culture. This result implies that of the 0.25 OD₆₀₀ in co-culture, about 0.21 OD₆₀₀ was contributed by N. europaea and 0.05 OD₆₀₀ by N. winogradskyi, indicating that the N. europaea benefitted the most from the co-culture conditions and that N. winogradskyi did neither better nor worse. Compared to single culture, the transcriptome of N. europaea in co-culture showed that mRNA levels of 773 genes (30% of the total genome inventory) changed significantly. Genes whose transcripts were present at higher levels in the co-culture included the NH₃ transporter (amtB), ATP synthase and NADH dehydrogenase components, cytochromes, carbonic anhydrase and genes implicated in fatty acid biosynthesis. Genes with transcripts expressed at lower levels in the co-culture included genes encoding for nitrite reduction (nirK), flagella synthesis, iron receptors, carbon dioxide fixation and other carbon assimilatory genes, and genes implicated in stress responses (glutathione synthesis, DNA repair and oxidative stress e.g. superoxide dismutase). Both growth and transcriptomic data show that N. europaea benefits from the interaction with N. winogradskyi in co-culture.
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