© Copyright 2016 Wei Qin Ecophysiology of marine ammonia-oxidizing archaea Wei Qin A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2016 Reading Committee: David A. Stahl, Chair Anitra E. Ingalls Robert M. Morris Program Authorized to Offer Degree: Department of Civil and Environmental Engineering University of Washington Abstract Ecophysiology of marine ammonia-oxidizing archaea Wei Qin Chair of the Supervisory Committee: Professor David A. Stahl Department of Civil and Environmental Engineering Ammonia oxidizing archaea (AOA) are one of the most abundant prokaryotes in the ocean and span diverse oceanic provinces. In addition to having a dominant role in marine nitrification, they are implicated as a major source of atmospherically active gases methane and nitrogen oxides. However, the scarcity of cultured isolates for laboratory study has hindered developing an understanding of specific metabolic traits and physicochemical factors controlling their activities and distribution. In this thesis, I report the isolation and characterization of three new marine AOA (strains HCA1, HCE1, and PS0) and show distinct adaptations to pH, salinity, temperature, light, and reactive oxygen species relative to the model AOA Nitrosopumilus maritimus strain SCM1. Increases in nitrous oxide (N2O) production in response to decreasing oxygen (O2) tensions was quantified and found consistent with an AOA contribution to the accumulation of N2O in suboxic regions of oxygen minimum zones. Normal growth of strain SCM1 was shown to be coupled with balanced production and consumption of nitric oxide (NO). A central role of NO in archaeal ammonia oxidation was confirmed by specific inhibition using an NO-scavenger (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide). The determination of high cellular quotas of cobalamin now implicates the AOA as major contributors to cobalamin in seawater. Gene expression studies showed that the entire cobalamin biosynthesis pathway is regulated by the level of nitrosative stress, suggesting that an interplay between NO production and cobalamin synthesis is central to the ecophysiology of marine AOA. Apart from having a major influence on the nitrogen cycle, their glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids are widely used to reconstruct past sea surface temperatures by means of TEX86 paleothermometer. However, the TEX86 proxy must now be reevaluated in consideration of the observation that O2 concentration greatly influences GDGT composition, leading to significant increases in TEX86-derived temperatures with increasing O2 limitation. Page List of Figures ................................................................................................................................ iii List of Tables ................................................................................................................................ vii Chapter 1. Introduction ................................................................................................................... 1 Chapter 2. Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation ................................................................................................16 Chapter 3. Nitrosopumilus maritimus gen. nov., sp. nov., Nitrosopumilus cobalaminogenes gen. nov., sp. nov., Nitrosopumilus oxyclinae gen. nov., sp. nov., and Nitrosopumilus ureaphilus gen. nov., sp. nov., four marine ammonia-oxidizing archaea of the phylum Thaumarchaeota......................................................................53 Chapter 4. Influence of oxygen availability on the activities of ammonia-oxidizing archaea ....102 Chapter 5. The production of nitric oxide by marine ammonia-oxidizing archaea and inhibition of archaeal ammonia oxidation by a nitric oxide scavenger .....................124 Chapter 6. Influence of ammonia and copper availabilities on the molecular physiology of the marine ammonia-oxidizing archaeon Nitrosopumilus maritimus ..............................164 Chapter 7. Confounding effects of oxygen and temperature on the TEX86 signature of marine thaumarchaeota ..........................................................................................................221 Chapter 8. Summary and Perspectives........................................................................................ 263 Chapter 9. Other Scientific Contributions .................................................................................. 269 i 9.1 Order Nitropumilales, Family Nitrosopumilaceae, Genus Nitrosopumilus ................ 269 9.2 Order Nitrosocaldales, Family Nitrosocaldaceae, Genus Nitrosocaldus ................... 271 9.3 Two distinct pools of B12 analogs reveal community interdependencies in the ocean 273 9.4 Ammonia oxidation kinetics and temperature sensitivity of a natural marine community dominated by Archaea ..............................................................................275 9.5 Variable influence of light and temperature upon ammonia oxidation activity in marine Thaumarchaeota ..............................................................................................277 ii Figure Number Page Figure 2.1. Growth and morphology of strains HCA1 and PS0. ..................................... 43 Figure 2.2. Phylogenetic relationships among amoA gene sequences of strains HCA1, PS0, and SCM1 ......................................................................................................... 44 Figure 2.3. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences. ................................................................................................................. 45 Figure 2.4. Temperature dependence of the growth of SCM1, HCA1, and PS0. ............ 46 Figure 2.5. Influence of pH and salinity on growth. ........................................................ 47 Figure 2.6. Photoinhibition and recovery of ammonia oxidation of the three AOA strains. ....................................................................................................................... 48 Figure 2.7. Nitrite production and growth curves of strains HCA1, PS0, and SCM1 with 100 µM α-ketoglutaric acid .............................................................................. 49 Figure 2.8. The specific growth yields of cultures of three AOA isolates with 100 µM α-ketoglutaric acid. ................................................................................................... 50 Figure 2.9. Growth of PS0 in artificial seawater medium containing urea...................... 51 Figure 3.1. Correlation of the growth of strain HCE1 with the stoichiometric ammonia oxidation to nitrite. .................................................................................................... 86 Figure 3.2. Influence of temperature on growth. ............................................................. 87 Figure 3.3. The effect of pH and salinity on the growth of strain HCE1......................... 88 Figure 3.4. Nitrite production by marine AOA isolates at different initial ammonia concentrations. .......................................................................................................... 89 Figure 3.5. Effect of organic compounds on the specific growth rate of strain SCM1. .. 90 Figure 3.6. The specific growth rates of strains HCA1, HCE1, and PS0 with supplementation of small organic acids .................................................................... 91 Figure 3.7. Correlation of the growth of strain SCM1 and total cobalamin synthesis. ... 92 Figure 3.8. Transmission electron micrographs of negative stained strain HCE1 cells. 93 Figure 3.9. S-layer structure of strain SCM1. .................................................................. 94 iii Figure 3.10. Maximum-likelihood tree based on the 16S rRNA sequences showing the phylogenetic relationships of the order Nitrosopumilales, the family Nitrosopumilaceae, and the genus Nitrosopumilus. ................................................. 95 Figure 3.11. Maximum-likelihood phylogenetic tree based on amoA gene sequences showing the phylogenetic relationships of Nitrosopumilus genus, Nitrosopumilaceae family, and Nitrosopumilales order. .......................................... 96 Figure 4.1. Oxygen-dependent growth kinetics of marine AOA. .................................. 116 Figure 4.2. Growth curve of strain HCA1 with α-ketoglutarate or H2O2 scavengers .... 117 Figure 4.3. The specific growth rates and specific growth yields of strain PS0 cultures supplemented with H2O2 scavengers, α-keto acids, or a common algal exudate. .. 118 Figure 4.4. Short-term and long-term effects of different concentrations of H2O2 on the ammonia oxidation activity of exponential phase SCM1 cells. ........................ 119 Figure 4.5. Growth curve of strain HCA1 with α-ketoglutarate or H2O2 scavengers. 120 Figure 4.6. Nitrite-normalized N2O yields of strain SCM1 with α-ketoglutarate relative to controls without α-ketoglutarate. ........................................................... 121 Figure 5.1. Metabolism of NO in SCM1. ...................................................................... 153 Figure 5.2. Growth of SCM1 as a function of initial ammonia and nitrite concentrations. ........................................................................................................ 154 Figure 5.3. Inhibition of ammonia