MIAMI UNIVERSITY

THE GRADUATE SCHOOL

CERTIFICATE FOR APPROVING THE DISSERTATION

We hereby approve the Dissertation of

Christopher James Sedlacek

Candidate for the Degree: Doctor of Philosophy

______Dr. Annette Bollmann, Director

______Dr. Rachael M. Morgan-Kiss, Reader

______Dr. Donald J. Ferguson, Reader

______Dr. Xiao-Wen Cheng

______Dr. Melany C. Fisk Graduate School Representative ABSTRACT

THE ECOPHYSIOLOGY OF NITROSOMONAS SP. IS79

by Christopher James Sedlacek

Nitrification, the two-step microbially mediated process of transforming ammonia

- (NH3) to nitrate (NO3 ) plays a large role in cycling nitrogenous compounds within and between both terrestrial and aquatic ecosystems. The first step in nitrification, ammonia oxidation, is carried out by ammonia-oxidizing microorganisms (AOM), both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). The study presented here focuses on the adaptation of AOB to different environmental conditions and their interactions with nitrite- oxidizing and heterotrophic bacterial community members. In order to investigate AOB species

+ adaptations to different environmental conditions, such as low ammonium (NH4 ) concentrations, cultivation dependent physiological growth experiments and whole genomic sequencing techniques were utilized. Comparison of whole and draft AOB genome sequences were used to correlate the presence or absence of genomic inventory with observed physiologic growth adaptations. The ability to identify and link genomic inventory with observed physiological adaptations allows for improved modeling of how changing environmental conditions will affect microbial community succession and overall ecosystem function. To investigate interactions between bacterial nitrifying community members, chemostat co-culture and community culture growth experiments were conducted with the AOB, Nitrosomonas sp. Is79; the nitrite-oxidizing bacteria (NOB), Nitrobacter winogradskyi and the enrichment culture G5-7. An isobaric tag for relative quantification (iTRAQ) proteomics approach was used to determine how the abundance of Nitrosomonas sp. Is79 proteins changed when grown in the presence of N. winogradskyi, heterotrophic bacteria or both in the enrichment culture G5-7. The growth rate of Nitrosomonas sp. Is79 increased when grown in co-culture with a N. winogradskyi or heterotrophic bacteria, but differential proteome shifts were observed. These differential proteome shifts produced a synergistic effect when Nitrosomonas sp. Is79 is grown in the presence of both N. winogradskyi and heterotrophic bacteria in G5-7. This synergistic effect involves abundance shifts in proteins involved in cellular pathways such as ammonia oxidation, the oxidative stress response and amino acid synthesis. Together these results highlight the importance of understanding species level adaptations within functional groups of microorganisms, such as ammonia oxidizers, and how co-culture or community level growth experiments can provide additional insights into microbial physiological characteristics.

THE ECOPHYSIOLOGY OF NITROSOMONAS SP. IS79

A Dissertation

Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology

by

Christopher James Sedlacek Miami University Oxford, OH 2015

Dissertation Director: Annette Bollmann, Ph.D.

TABLE OF CONTENTS List of Tables iii List of Figures v Acknowledgements vi Introduction 1 Chapter 1. Physiological and Genomic Comparison of Ammonia-oxidizing Bacteria Adapted to Different Ammonium Concentrations 23 Chapter 2. The effect of bacterial community members on the proteome of the ammonia-oxidizing bacterium Nitrosomonas sp. Is79 53 Summary 100 References 110

ii" LIST OF TABLES Table Page 1. AOB isolation environment and Nitrosomonas cluster affiliation 28 2. General chracteristics of Nitrosomonas cluster 6a and 7 AOB genomes 37 3. tRNA genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes 38 4. Ammonia oxidation related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes 40 5. Terminal oxidase genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes 42 6. Urea and hydrogen utilization related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes 44 7. Nitrogen oxide metabolism related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes 47 8. Carbon metabolism related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes 50 9. 16S rRNA bacterial primers used for amplification and sequencing 61 10. Phylogenetic affiliation of the heterotrophic bacteria present in the enrichment culture G5-7 67 11. Phylogenetic affiliation of the bacterial isolates, isolated from the enrichment culture G5-7 68 12. Half saturation constant of ammonia-oxidizing activity and growth of Nitrosomonas sp. Is79 in pure culture, in the presence of N. winogradskyi and as part of the enrichment culture G5-7 71

+ - - 13. Steady state NH4 , NO2 and NO3 concentrations in the continuous cultures: Nitrosomonas sp. Is79, Nitrosomonas sp. Is79 and N. winogradskyi and the enrichment culture G5-7 77 14. Nitrosomonas sp. Is79 proteins that were detected when in co-culture with N. winogradskyi compared to when grown as a pure culture 80 15. Nitrosomonas sp. Is79 proteins that were detected when grown as part of the enrichment culture G5-7 compared to when grown as a pure culture 85

iii" 16. Nitrosomonas sp. Is79 proteins that changed in abundance when in co-culture with N. winogradskyi or grown as part of G5-7 compared to when grown as a pure culture 90

iv" LIST OF FIGURES Figure Page 1. Simplified model of the nitrogen cycle 4 2. Simplified model of energy and reductant generation in AOB 11 3. Neighbor-joining tree of AOB based on 16S rRNA gene nucleotide sequences 33

+ - 4. The influence of initial NH4 concentration, NO2 concentration, pH,

headspace O2 concentration and urea on the growth rate of AOB 35 - - - 5. NO2 or NO2 /NO3 production by Nitrosomonas sp. Is79, Nitrosomonas sp. Is79 + N. winogradskyi, and G5-7 70 6. Growth rate of Nitrosomonas sp. Is79 in co-cultures and community cultures 73

+ - - 7. NH4 , NO2 and NO3 concentration in continuous cultures of Nitrosomonas sp. Is79, Nitrosomonas sp. Is79 co-cultured with N. winogradskyi and the enrichment culture G5-7 76 8. SDS-PAGE of whole cell extracts from Nitrosomonas sp. Is79, Nitrosomonas sp. Is79 co-cultured with N. winogradskyi, and the enrichment culture G5-7 grown to steady state in continuous culture 79 9. Model of Nitrosomonas sp. Is79 proteome shifts in response to being grown in the presence of N. winogradskyi, heterotrophic bacteria or as part of the enrichment culture G5-7 106

v" ACKNOWLEDGEMENTS First and foremost I would like to thank my mentor Annette. Without her support, guidance and patience I never would have acquired the skills I have now or become the scientist I am today. When I joined the Bollmann lab, my only condition was that I would never work on the ammonia-oxidizers. Following these acknowledgements is my dissertation solely based on characterizing ammonia-oxidizing bacteria. As I look back now, I never could have imagined the relationships, mentorships and friendships that all started because “culturing the uncultureables” and uranium bioremediation sounded so fascinating. Secondly, I would like to acknowledge all the help I received along the way from my committee members: Rachael, Melany, DJ and Xiao-wen. They stayed with me through all the 4pm Friday committee meetings, listened when I would bank my whole dissertation on a single ill-fated idea and always kept me thinking critically with their advice. I would especially like to thank Rachael for giving me the unbelievable opportunity to join her Antarctic field research team; it was an experience and field season like no other (B-247). Without my family I never would have made it to graduate school. Never give up, work hard, and most importantly never lose yourself. That is what I learned from my family and that is what got me to where I am today. Thanks Mom, Dad, Jess and Jon for all your support. Throughout the years in the Bollmann lab, you probably couldn’t have assembled a weirder more eccentric group of people if you tried. There were lots of great undergrads I was able to work with, including Austin, Rhea and Britton but I would like to single out Brian McGowan. Brian is a great researcher and an even better person. I thank him for all his help in the lab and his continued friendship outside of the lab. Broomball champions, Cabrewing mates, Camping veterans, Team Steve, Plain blank tees; by the end of so many years we could call it by a lot of different names but it boils down to friends. I’ve made more than a few lifelong friends in the past six years and appreciate all the time we spent together both inside and outside of the Pearson Hall basement. As a founding member of OVUM, I hope the needle never stops spinning here in Oxford.

-Cheers

vi" INTRODUCTION Microorganisms in the environment are subjected to gradients of abiotic factors that are in constant flux. Environmental conditions such as substrate concentration, substrate availability, pH, temperature, light, and oxygen concentration play large roles in determining the diversity of microorganisms that can survive in a particular habitat. Freshwater lakes are an example of an environment with constantly varying conditions and spatially diverse habitats. In order to understand how microorganisms are adapted to different environmental habitats, genomic comparisons in conjunction with laboratory growth experiments are frequently used. For example, this type of combined approach has been utilized to differentiate adaptations in ammonia-oxidizing bacteria (AOB) (1, 2), Bacillus species (3) and Sulfobacillus species (4). In natural habitats, abiotic conditions are not the only factors influencing microbial growth: microorganisms function as part of intricate, interacting, and often complex communities. These interactions can range from positive interactions such as quorum sensing (5, 6) or cross feeding (7-10), to negative interactions such as competition or predation (11, 12). Proteomics based approaches have been used to investigate these types of interactions within a wide range of naturally occurring microbial communities including: acid mine drainage biofilms (13, 14), biostimulated sediment (15), and marine environments (16). A similar approach has also been applied to interactions within enriched or artificial communities involved in methanogenesis (17) and the ability of microbial communities to, for example, produce Vitamin C precursors (18), withstand antibiotic treatment (19) or tolerate chemical/oxidative stress (20). Determining how microorganisms are adapted to their environment and interact with community members is essential to understand how microbial succession in a community will affect ecosystem processes.

The Nitrogen Cycle Nitrogen is an essential nutrient for life, as it is a component of crucial biomolecules including amino acids for protein production and nucleic acids for DNA and RNA molecules.

Dinitrogen gas (N2) is the most common form of nitrogen in the environment. It makes up roughly 79% of the atmosphere, which makes the atmosphere the largest natural reservoir of nitrogen. Although very abundant, N2 is not biologically available for use by most organisms.

For nitrogen to become biologically available, N2 must be fixed into ammonia (NH3) /

" 1" + ammonium (NH4 ) through nitrogen fixation. This process connects the atmospheric and biospheric reservoirs of nitrogen based compounds (Fig. 1). Select bacteria (21) and archaea (22), called diazotrophs, are capable of nitrogen fixation. Diazotrophs can be found as free-living cells such as cyanobacteria (23) or in symbiotic relationships within root nodules of leguminous plants (24). To fix nitrogen, all diazotrophs utilize a nitrogenase enzyme complex (21, 25). Nitrogenase enzymes are extremely oxygen sensitive, due to exposed iron-sulfur clusters on their surface (21). Diazotrophs possess several different physiological strategies to limit the exposure of nitrogenase enzyme complexes to oxygen, including: oxygen consumption through respiration, compartmentalization of nitrogenase enzymes or an anaerobic lifestyle.

+ Once fixed into the biologically available form of NH3 or NH4 , nitrogen can be assimilated into organic biomolecules or used for energy generation by chemolithoautotrophic microorganisms. The latter occurs during the next step in the nitrogen cycle: nitrification. Aerobic nitrification is a two-step process that involves the transformation of the most reduced

+ - form of nitrogen (NH4 ), to the most oxidized form (nitrate (NO3 ) (26). The first and rate- - limiting step, ammonia oxidation, is the oxidation of NH3 to nitrite (NO2 ). The second step, - - nitrite oxidation, is the subsequent oxidation of NO2 to NO3 (Fig. 1) (27, 28). The two steps of - nitrification co-occur in the environment, which often leaves undetectable levels of NO2 , and - - - therefore NO3 as the only measurable end product of nitrification (29). Both NO2 and NO3 are biologically available forms of nitrogen, which can be assimilated by plants or other microorganisms. Various heterotrophic bacteria, fungi and algae (30-32) are capable of

- aerobically oxidizing NH3 to NO3 , through a process called heterotrophic nitrification (33, 34). Heterotrophic nitrification is not coupled to energy generation in these microorganisms and is considered to contribute to local nitrification rates but only play a minimal role in the global nitrogen cycle (35, 36). Although traditionally thought to be a strictly aerobic process, ammonia oxidation can occur anaerobically through a process called anammox (ANaerobic AMMonia OXidation) (37,

- - 38). During anammox NH3 is oxidized and NO2 is reduced, forming N2 and NO3 as end products. This process is carried out by a deep branching group of monophyletic microorganisms within the phylum Planctomycetes (37, 38). Anammox bacteria possess a specialized organelle, the anammoxosome, which is the only place in the cell that the anammox reaction occurs (39, 40).

" 2"

Figure 1. Simplified model of the nitrogen cycle. Major microbially mediated processes within the nitrogen cycle are: nitrogen fixation, nitrification, denitrification and anammox. Ammonification can occur through microbial or chemical / oxidative mediated processes.

" 3"

Nitrification - NO2 Ammonia Nitrogen Fixation Oxidation Organic N Nitrite Nitrogen Oxidation 2 - NO3 Ammonification Oxic NH /NH + Anoxic 3 4 Annamox - NO2 Nitrogen Fixation

N2 NO

N2O Denitrification

" 4" - Classical aerobic nitrification, heterotrophic nitrification and anammox all produce NO3 - as a soluble end product. NO3 is utilized anaerobically as a terminal electron acceptor and is reduced back to N2 through a process called denitrification, which closes the nitrogen cycle (Fig. - - 1). Denitrification involves the successive reduction of NO3 to NO2 , nitric oxide (NO), nitrous oxide (N2O) and lastly N2 (41). The ability of microorganisms to perform partial or full denitrification is widespread due to a large number of lateral gene transfers (42, 43). Less

- + commonly, NO3 can also be reduced directly to NH4 through a process called dissimilatory nitrate reduction to ammonium (DNRA) (41, 44). DNRA, unlike denitrification, keeps nitrogen in the biosphere where it is still accessible to microorganisms. The process of organic

+ + decomposition or ammonification also adds to the amount of free NH4 in the biosphere as NH4 is released from the breakdown of larger organic nitrogen containing molecules, such as nucleic

+ - acids and proteins (45). These terrestrial and aquatic pools of NH4 and NO3 are used by + - microorganisms that cannot fix N2 directly but can assimilate NH4 or NO3 into organic cellular biomolecules through a process called nitrogen assimilation (46, 47).

Environmental Impact of the Nitrogen Cycle

- Nitrification provides biologically available nitrogen in the form of NO3 , the most - abundant biologically available form of nitrogen in the environment. Although, both NO3 and + NH4 can be utilized as a nitrogen source by plants and most photosynthetic microorganisms, it - + + requires more energy to uptake and intracellularly reduce NO3 to NH4 than to uptake NH4 directly (48, 49). However, nitrogen is a limiting nutrient in many environments including many freshwater, marine and soil environments (50, 51). With the invention of the Haber-Bosch

th process in the early 20 century, which produces NH3 from N2 and hydrogen, humans have had the means to add anthropogenic nitrogen to the environment on a large scale (52, 53). Currently about 150 Tg of anthropogenic nitrogen is added to the environment each year (54). Most of

+ which is in the form of NH4 or urea in fertilizers that are applied to terrestrial agricultural environments. This input of anthropogenic nitrogen into the biosphere has caused an unbalancing of the nitrogen cycle, which is linked to a large number of environmental issues (53, 55). The main source of nitrogen based environmental issues is caused by the fact that anthropogenic nitrogen is not confined to the site it is applied and our understanding of to the fate of applied nitrogen is limited (56-58). Soil microorganisms involved in nitrification not only

" 5" + - compete with crops for applied NH4 , but also produce NO3 as a soluble end product. Due to its - charge, NO3 , has negligible interactions with the negatively charged matrix of topsoil and is + therefore much more susceptible to both leaching and run-off than NH4 (59, 60). Although crops - can take up NO3 as a biologically available source of nitrogen, it is more prone to being removed from the soil (59, 60). Leaching and run-off processes transport nitrogen compounds from soil

- into aquatic ecosystems. In an aquatic ecosystem, excess nitrogen compounds like NO3 play a large role in eutrophication, as many freshwater and marine environments are nitrogen limited (61, 62). Influxes of nitrogen along with phosphorous compounds have been previously shown to be stimulating factors of increasing phytoplankton productivity and harmful algal blooms (HABs) (63, 64). HABs have a detrimental impact on the overall health of aquatic ecosystems by lowering the biotic diversity of the ecosystem, triggering zones of hypoxia within the water column and producing toxins (63-66). These issues can also have an economical impact on commerce, depending on the organisms affected in these ecosystems (53, 54, 63). In addition, nitrifying microorganisms found in both soil and aquatic environments have been shown to aerobically produce nitrogen oxides including N2O (67), which is a potent greenhouse gas

(GHG) with 300 times the global warming potential of carbon dioxide (CO2) (68). During aerobic nitrification, N2O is most likely produced through the enzymatic reduction of NO from the incomplete oxidation of hydroxylamine (NH2OH) (69, 70). In contrast, during periods of - reduced oxygen (O2) tension or anoxia, nitrifiers can reduce NO2 to N2O through a process called nitrifier denitrification (69-72). Denitrifying microorganisms can also produce N2O and - other nitrogen oxides such as NO through incomplete denitrification of NO3 to N2 (71, 73). Taken together, excess fixed nitrogen in the environment is linked to: increased nutrient runoff from agricultural environments, the increasing number of HABs (74), and the rising concentration of nitrogen oxides in the atmosphere (75). These are some of the reasons that the National Academy of Engineering named managing the nitrogen cycle one of the top fourteen grand challenges of the 21st century (National Academy of Engineering, 2008). The ability to regulate the processes involved in the nitrogen cycle, especially nitrification, has great value in natural and engineered environments. Controlling and promoting nitrification in wastewater treatment plants (WWTPs) is essential for dealing with human waste

+ because NH4 and urea are the most common forms of nitrogen-based compounds found in human wastewater and sewage. Before wastewater is put back into waterways the amount of

" 6" nitrogen-based compounds are reduced through nitrification and subsequent denitrification, releasing nitrogen into the atmosphere as N2 (76, 77). Without the ability to control these processes in an engineered environment, human waste would be play a large role in the eutrophication of natural environments.

Freshwater Environments Freshwater lakes make up a small fraction of the total water on earth, but contain the majority of available non-frozen freshwater and are the largest source of drinking water in the world (78). Freshwater lakes are fed by surrounding watershed areas, which deliver water and nutrients to lakes through runoff and streams (78). The land use in the surrounding watershed area of a lake impacts the amount of nutrients the lake receives. Most of the nutrient inputs that cause environmental issues come from watershed areas composed of mainly agricultural fields, where fertilizers containing high amounts of nitrogen or phosphorus compounds have been applied. Freshwater lakes can have varying trophic states from ultraoligotrophic to hypereutrophic depending on their level of biologically available nutrients. Eutrophic lakes are characterized as having high nutrient content, high primary productivity, murky waters and a shallow oxic zone. On the other end of the spectrum, oligotrophic lakes are characterized as having low nutrient content, low primary productivity, clear water with high visibility and oxygenated zones that extend deep into the lake as far as the bottom (79). Oligotrophic lakes are more sensitive to nutrient inputs because a relatively small nutrient input can largely affect the total nutrient load in the system. Nitrification in freshwater lakes has been well studied for many years (80). The two

+ substrates of nitrification, NH4 and O2, are most abundant on opposing ends of the vertical + stratification of freshwater lakes. O2 is most abundant at the surface while NH4 is most abundant in the sediment. The majority of nitrification occurs within the top layer of sediment, but this can vary based on the lake and time of the year (81). Historically, cultivation dependent methods were used to characterize freshwater nitrifiers but this is a time intensive process due to the overall slow growth of nitrifying microorganisms. Now, cultivation independent methods are favored in the characterization of nitrifying populations (82, 83). These molecular methods allow for a much better understanding of the diversity and distribution of the nitrifiers present (84-87),

" 7" but may not give an accurate picture of which nitrifiers are actively growing or oxidizing NH3 (88).

The Study of Nitrifying Microorganisms Nitrifying microorganisms have been studied since being discovered by Sergei Winogradsky over 120 years ago (89, 90). Winogradsky was the first to describe the chemolithoautotrophic lifestyle of both AOB and nitrite-oxidizing bacteria (NOB) (90). Along with Robert Warrington, they showed that AOB and NOB each complete one step of nitrification independently (90, 91). These discoveries directly led to the idea of biogeochemical cycles (92). Until 2004, it was believed that AOB and NOB were the only major players in aerobic nitrification. This ideology changed when two separate research groups found ammonia- oxidation specific genes on genetic scaffolds belonging to the phylum Crenarchaeota (93, 94). The controversy was put to rest when the first ammonia-oxidizing archaea (AOA), Nitrosopumilus maritimus was isolated in 2005 (95). Since then only two other AOA species, Nitrososphaera viennensis (96) and Nitrosotalea devanaterra have been isolated (97, 98). However, AOA enrichment cultures have also been reported from freshwater (88), estuarine (99), agricultural (100) and oceanic environments (101). AOB and AOA share their

- chemolithoautotrophic metabolism, as they both oxidize NH3 to NO2 to generate their cellular energy and reducing power (102). They also often co-occur in the same environments, which have led to open eco-physiological and biochemical questions about their growth, activity and their niche differentiation (88, 102-104). The similarities between AOB and AOA end with their general modes of chemolithoautotrophic metabolism. Although both fix CO2, AOB utilize the Calvin cycle (105) and the AOA use a modified 3-hydroxypropionate/4-hydroxybutyrate pathway (102, 106). In addition, the ability to isolate AOA cultures has relied on the addition of seemingly species- specific carbon compounds to the typical inorganic ammonia-oxidizer medium used to isolate AOB. Due to the relatively recent discovery of the AOA, many components of their biochemistry have yet to be elucidated and therefore direct comparisons with AOB is in its infancy. Examples of open questions regarding AOA vs. AOB metabolism include: production of NO or N2O intermediates, the specific intermediate compounds produced during ammonia oxidation, and the relative contribution of AOA to the overall ammonia oxidation in different

" 8" environments. The latter has been under extensive study since the discovery of AOA but studies have mainly utilized molecular methods, which have been used to determine the number of AOB or AOA present in a given environment (107-109). Population numbers have then been used to estimate whether AOA or AOB are responsible for oxidizing more NH3 in particular environments. However, caution must be used when estimating ammonia-oxidizing activity based on cell number or even 16S rRNA copies, as AOA and AOB have been shown to retain 16S rRNA for months without substrate (110). Future culture-based biochemical and competition studies will continue to shed light on the niche differentiation between the AOB and the AOA.

Ammonia-oxidizing Bacteria The majority of the biochemical, physiological and genetic characterization of AOB has focused on a single model organism, Nitrosomonas europaea. The emphasis on this AOB as a model species is because N. europaea has a relatively fast generation time of about 7 hours (111)

+ and can be grown in the presence of high NH4 concentrations of up to 100mM; which provides the necessary biomass for biochemical assays. Select few AOB can be grown on solid agar plates, one of which is N. europaea, which has allowed for the development of genetic tools (112). Most

+ AOB can not grow at such high environmentally irrelevant NH4 concentrations. Until recently, + AOB adapted to low NH4 environments have largely been ignored in both cultivation and physiological experiments, because of their slow growth rate and their low biomass production.

+ A better understanding of AOB species adapted to low NH4 environments is necessary to understand how nitrifying communities are being affected by anthropogenic influences that are causing eutrophication in previously oligotrophic aquatic environments worldwide.

Energy generation and carbon fixation AOB have a chemolithoautotrophic metabolism, where cellular energy and reducing

- power are generated solely from the oxidation of NH3 to NO2 . The oxidation of NH3 is carried out as a two-step process, which involves the oxidation of NH3 to NH2OH (113) by the membrane bound ammonia monooxygenase (Amo) complex (114) (Fig. 2). The Amo complex consists of three dimerized subunits: AmoA which contains the active site of the enzyme complex, AmoB and AmoC, which are all part of the genetically encoded amo operon (115). All AOB sequenced to date except Nitrosococcus species have more then one copy of the amo

" 9"

Figure 2. Simplified model of energy and reductant generation in AOB. AMO, ammonia monooxygenase; HAO, hydroxylamine dehydrogenase; c554, cytochrome c554; cm552, cytochrome cm552; Q/QH2, ubiquinone-ubiquinol pool; bc1, cytochrome bc1 (complex III); c552, cytochrome c552; HCO (c)aa3, cytochrome (c)aa3; ATP Syn, ATP synthase; NADH DH, NADH dehydrogenase; PMF, proton-motive force.

" 10"

HAO 4e- c554

Periplasm NH 3 - NH OH NO2 + PMF c552 + 1/2O2 2 PMF - PMF 2e- 2e PMF PMF cm552 - AMO 4e HCO ATP 2e- NADH 2e- 2e- bc1 Syn (c)aa3 DH Q/QH2 Q/QH2

2H+ H+ H+ Cytoplasm H+ ADP ATP + 1/2O2 H2O NAD NADH + 2H+

" 11" operon in their genome (2, 116-120). It is likely that these multiple operon copies originated from relatively recent gene duplication events (121). Previous studies have shown evidence for differential regulation of the operons at the transcriptional (122), translational (123, 124) and post-translational levels (124); however, no single amo operon in any AOB has been shown to be essential for cellular growth (125). During the second step of ammonia oxidation, the

- intermediate NH2OH is further oxidized to NO2 by the periplasmic hydroxylamine oxidoreductase (Hao) complex (126) (Fig. 2). Similar to the amo operon, the hao genes are found in a gene cluster and are often found in multiple non-essential operon copies within AOB genomes (112).

The oxidation of one molecule of NH3 generates four electrons (127) but nets AOB only - two. Of the four electrons generated by the oxidation of NH2OH to NO2 , two are recycled to Amo to prime the enzyme complex (128) and two are shuttled through cytochrome c554 and cytochrome cm552 to the ubiquinone pool (126). These two electrons are used for both cellular respiration and reverse electron flow. Electrons that pass to the terminal oxidase, cytochrome aa3, are respired and create proton-motive force (PMF) (129) used in ATP and NADH production (130, 131). Some electrons enter directly into reverse electron flow for NADH production (131) (Fig. 2). In silico it appears that reverse electron flow in AOB is ATP dependent (132); however it is hypothesized that in vivo, reverse electron flow may be ATP independent and rely solely on PMF, which has been shown for the NOB (133). The ability to use PMF instead of ATP to power reverse electron flow decouples the need to first generate ATP in order to produce NADH or NAD(P)H.

AOB acquire carbon for cellular growth by autotrophically fixing CO2 through the Calvin cycle (134). Depending on natural species-specific environmental adaptations, AOB have different methods of maximizing CO2 fixation. The ability to produce carboxysomes or genetically encode multiple forms of ribulose-1,5-Bisphosphate carboxylase/oxygenase (RuBisCO) enzymes are both strategies utilized by AOB species (1, 2, 119). Carbon fixation is an energy intensive process, requiring both ATP and NAD(P)H. It has been estimated that AOB spend about 80% of their cellular energy on carbon fixation (135). Nitrosomonas eutropha and N. europaea have been shown to grow chemoheterotrophically and assimilate small amounts of organic carbon based sugars, such as fructose or pyruvate, under anoxic conditions (136). The

" 12" uptake of organic molecules by AOB is limited by the small genetic inventory of organic transporters AOB species possess (1, 2, 119).

Phylogeny, ecophysiology and genetics Historically AOB were classified by cellular morphology and assigned to one of five genera (137). With the advent of 16S rRNA based phylogeny, the number of genera has been reduced to three: Nitrosomonas, Nitrosospiria and Nitrosococcus (138). To date all sequenced AOB belong to one of two monophyletic linages within either the β- or γ-Proteobacteria (139). The γ-Proteobacterial AOB all belong to the genus Nitrosococcus, which contains two recognized species, Nitrosococcus oceani and Nitrosococcus halophilus. Both species were isolated from and are routinely detected in marine environments around the world (140). Within the β-Proteobacterial AOB, there are 16 named, isolated and described species that belong to either the Nitrosospira or Nitrosomonas genera. Based on 16S rRNA gene sequences Nitrosospira, species cluster into one of five phylogenetic lineages that correspond to the abiotic environmental conditions of their habitat: environments that have minimal exposure to anthropogenic inputs (clusters 0, 3 and 4), marine environments (cluster 1) and acidic soil environments (cluster 2) (141). The phylogenetic clusters within Nitrosospira are not all supported with cultured representatives, which makes deeper ecophysiological comparisons between the different clusters difficult. Conversely, Nitrosomonas has a substructure of six distinct clusters based on 16S rRNA sequencing that are all represented by at least one isolated species. The major physiologic characteristics that separate the six lineages with the genus Nitrosomonas are substrate affinity (140, 142) and salt tolerance (80). The N. europaea cluster contains four described species N. europaea, N. eutropha, Nitrosomonas mobilis and Nitrosomonas halophila; that were isolated and are often detected in both soil and aquatic in

+ environments that have high free NH4 concentrations, like WWTPs, agricultural fields, or eutrophic lakes. Members of this cluster are urease negative and the fully sequenced members are missing the genetic inventory necessary to utilize urea (80). In contrast, members of the Nitrosomonas oligotropha cluster are commonly detected and isolated from soil and aquatic

+ environments with low NH4 concentrations like oligotrophic lakes or soils with little to no anthropogenic impact. Species within this cluster are usually urease positive, which is an adaptation for survival in oligotrophic environments because it liberates previously unavailable

" 13" + + NH4 in a low NH4 environment (80). Currently there are two described members within the N. oligotropha cluster: N. oligotropha and Nitrosomonas ureae, but there are several more species in this cluster in the process of being physiologically and genomically characterized (Sedlacek unpublished). The species within the N. oligotropha cluster that is currently being characterized and is the focus of the study presented here is Nitrosomonas sp. Is79. Nitrosomonas sp. Is79 was originally studied as a member of the ammonia-oxidizing enrichment culture G5-7, which was enriched from fresh water sediment in lake Drontermeer in The Netherlands (52o58’N, 5o50’E) (143). A low ammonium continuous culture technique was used in the enrichment process. It has since been isolated, physiologically characterized (Sedlacek unpublished) and its genome has been fully sequenced and annotated (2). The axenic culture of Nitrosomonas sp. Is79 along with well characterized enrichment culture G5-7 create a good model system for investigating both species-specific adaptations and AOB community interactions because of the ability to study the physiology of Nitrosomonas sp. Is79 alone or in an environmentally relevant microbial community. To date there are 10 sequenced AOB species publicly available (1, 2, 116-120). Many of which have been sequenced very recently and their genomic makeups are currently under review. Together, the 10 species represent AOB with diverse phylogenetic backgrounds within all three genera of AOB: Nitrosococcus, Nitrosospira and several different clusters within Nitrosomonas. Further sequencing and genomic comparison of several AOB from different clusters within Nitrosomonas is currently ongoing to provide a clearer picture of the physiological differences between the different clusters and their genetic basis.

Nitrite-oxidizing Bacteria Historically, NOB have not been as well studied as AOB. The majority of NOB physiological and genetic studies have utilized Nitrobacter winogradskyi as a model organism because of its relatively high growth rate (144). Cultivation of NOB axenic cultures from the environment is tedious because like AOB; NOB do not readily grow on solid agar plates and have slow growth rates with generation times upwards of 10 hours in optimal conditions (145). In the environment, NOB are often found in close proximity or even in multispecies associations with ammonia-oxidizing microorganisms (AOM) (146). This close interaction with AOM is

" 14" mutually beneficial as the NOB are cross-fed by AOM and simultaneously remove growth inhibitory waste products produced by the AOM (7, 8, 147). When grown in co-culture with N. europaea, N. winogradskyi has been shown to differentially regulate transcription of 11.8% of its genome (147).

Energy generation and carbon fixation NOB are chemolithoautotrophs and complete the second step in nitrification, the

- - oxidation of NO2 to NO3 , to generate cellular energy and reducing power (126). The nitrite - - oxidoreductase (Nxr) enzyme complex oxidizes NO2 directly to NO3 . Nxr is a heterodimeric protein complex comprised of one NxrA subunit and one NxrB subunit (148). However, the exact location of the Nxr complex is a debated issue with hypotheses ranging from membrane bound on either the cytoplasmic side or the periplasmic side of the cell membrane to being unattached to a membrane and located in the periplasmic space (149, 150). Genomic sequencing has recently shown that most NOB have multiple copies of nxrA and nxrB gene clusters, but only one central operon which includes all the additional necessary accessory proteins (151). Electrons generated are passed from the Nxr complex to cytochrome c550 and ultimately to a terminal cytochrome oxidase (152) or are used in reverse electron flow (133). The slow growth rates of NOB are in part because of the low amount of energy generated from the oxidation of

- - NO2 balanced against the high energy requirements for CO2 fixation: ~100 mol of NO2 to fix 1 mol CO2 (153). Nitrite oxidation generates about half of the energy ammonia oxidation does (154). NOB can grow chemoorganotrophically (27), mixotrophically (27), or heterotrophically under specific laboratory conditions (27, 155) but the ecological occurrence and importance of these lifestyles are unknown.

NOB species utilize diverse mechanisms and pathways to capture CO2 needed for cellular autotrophic growth. Like AOB, the majority of cultured NOB fix CO2 via RuBisCO and the Calvin cycle, with certain species employing carboxysomes (156). However “Candidatus Nitrospira defluvii” is the first sequenced nitrifying microorganism to not possess the genes necessary for the complete Calvin cycle, but instead encodes multiple carbonic anhydrase genes (157).

Phylogeny and ecophysiology

" 15" - The ability for microorganisms to use NO2 as an energy source is found in a very diverse group of microorganisms which belong to five genera: Nitrobacter, Nitrococcus, Nitrospina, Nitrospira or the Candidate genus “Nitrotoga” that are each in a unique phylum: α- proteobacteria, γ-proteobacteria, δ-proteobacteria, Nitrospirae, or the β-proteobacteria, respectively. Although NOB share a core metabolism, the habitat selection and the closest relatives of these microorganisms varies greatly. The genera Nitrococcus and Nitrospina are each represented by just one described species of marine origin and are commonly found in the subeuphotic zone of the ocean along with AOA (158). The candidate genus “Nitrotoga” contains the first described cold adapted NOB species, “Candidatus Nitrotoga arctica”, which was

- o o enriched from permafrost soil in Siberia and is able to oxidize NO2 at 4 C but not at 25 C (159). Species from the two more heavily studied genera Nitrobacter and Nitrospira, are found more ubiquitously in a wide range of environments and dominate NOB populations in industrially applicable environments such as WWTPs. Species found within the genus Nitrobacter have an interestingly low genetic diversity on the 16S rRNA level, but are highly ecologically diverse and inhabit many distinctly different environments (160). Nitrospira species often co-occur with Nitrobacter species in the environment. The niche differentiation between Nitrobacter and Nitrospira species is the focus of many ongoing studies in both natural and engineered environments (161, 162). The NOB utilized in the study presented here is N. winogradskyi. N. winogradskyi has been previously physiologically characterized (163), has a fully sequenced genome (156), has been previously used in interaction studies with AOB (147), and is the only NOB within the environmental enrichment culture G5-7 (164). These characteristics allow N. winogradskyi to be used as a model NOB in investigating interactions between AOB and NOB community members

+ under NH4 -limited conditions.

Cultivation of Nitrifying Microorganisms Nitrifying microorganisms are characteristically difficult to cultivate in the laboratory because they do not readily grow on solid agar plates, produce a small amount of biomass, and often take weeks to grow. These limitations have led the field to generally avoid traditional cultivation methods and rely heavily on molecular methods such as the enumeration of nitrifier communities through quantitative polymerase chain reaction (qPCR) (165, 166) or metagenomic

" 16" sequencing (167). This shift away from cultivation and towards molecular techniques has allowed for more environments to be sampled and deeper branching phylogenetic trees to be made. However it does not allow for any ecophysiological or growth characterization experiments to be performed on any novel organisms discovered. Cultivation is necessary to thoroughly describe the full environmental biodiversity and to determine if predictions about sequenced environmental microorganisms are accurate (168). Controlled experimental manipulation of environmental conditions is essential in determining which factors drive the community structure of nitrifying microorganisms.

Enrichment and isolation Nitrifiers cannot be isolated directly from the environment onto solid agar plates due to their low biomass production and inability to form visible microcolonies. Therefore, enrichment cultures are utilized in place of more direct isolation techniques to increase the biomass of a desired group of nitrifiers. Typically, enrichment culture medium is composed of a general mineral salts medium supplemented with HEPES or a phosphate based buffer and an appropriate substrate (141, 169). The amount of substrate added can vary greatly depending on the environment the sample is from or the characteristics of the desired nitrifier. Enrichment culture media along with the amount of substrate added could select for particular species that may not necessarily represent the most abundant populations present in the natural community (170-172). An environmentally relevant level of substrate should be selected to increase the likelihood of enriching for the most dominate species in a particular environment. Enrichment cultures are monitored for substrate consumption and a portion of the enrichment culture is transferred to fresh medium when necessary. After several transfers the nitrifiers will be the most abundant microorganisms in the enrichment culture, although there will still be heterotrophic bacteria and possibly other nitrifying populations in the culture. Isolation of nitrifiers from enrichment cultures is usually a time-intensive technique because of their naturally slow growth rate and the fact that they often grow in multicellular flocs. A dilution-to-extinction method is used for nitrifier species; which involves serially diluting the enrichment culture into fresh medium (173). The serially diluted culture should be allowed to incubate for one to several months, at which point the most diluted culture that shows growth is selected to start the next round of the dilution-to-extinction method. This method is repeated

" 17" until a pure nitrifier culture is obtained. Contaminating bacteria can be detected by inoculating the culture into heterotrophic medium or by sequencing a clone library of the culture (169). In theory, isolating heterotrophic bacteria from a nitrifier enrichment culture is a much simpler task than isolating the nitrifier itself. Plating the enrichment culture onto several different types of solid agar medium and selecting single colonies if often enough to isolate the majority of heterotrophic bacterial species present. However, not all heterotrophic bacteria can be isolated through these methods. The isolated bacterial diversity can be compared against a 16S rRNA pyrosequencing profile or clone library of the microorganisms in the enrichment culture.

Batch cultures Enrichment cultures and axenic nitrifier stock cultures are often kept in batch culture indefinitely because not all nitrifier cultures reliably revive after being frozen or lyophilized with traditional methods developed for heterotrophic bacteria. However, investigation into novel long- term frozen storage techniques for these organisms is being conducted (174); however, current efforts involve the addition of carbon-based additives to the culture, which promotes contamination of axenic cultures upon revival. Batch cultures are monitored for substrate depletion as well as waste production and transferred to fresh medium when necessary. Stock batch cultures are often transferred at least once a month to ensure stable maintenance, even though starvation experiments have shown that certain nitrifiers can recover from long-term starvation (88, 110, 175).

Continuous cultures Continuous or chemostat cultures can be used in the original enrichment process or to further characterize established laboratory cultures (143, 147, 164, 169). The use of chemostats allows for many abiotic parameters to be kept constant during long-term microbial incubations. Abiotic parameters such as pH, oxygen concentration, and temperature can be constantly monitored through sensors within the chemostat and manipulated in real time through computer- controlled pumps. Fresh medium is added to the growth vessel at a constant rate and culture is removed at a constant rate to prevent the build-up of waste products or dead biomass. When growing at steady state, the growth rate of the culture is constant and is controlled by the dilution rate of the fresh medium supplied (169).

" 18" Culture-based ecophysiological experiments performed in chemostats have several advantages over similar experiments performed in batch cultures. The most important factor is the ability to monitor growth under constant and very reproducible conditions. In a growth experiment performed in a chemostat, once steady state growth is achieved, the substrate concentration, concentration of waste products and pH do not change over the course of the experiment as they would in a batch culture. Chemostats can also be used to produce greater quantities of microbial biomass, which is needed for reliable proteomic or transcriptomic experiments. This allows for large volumes of culture to be grown under constant conditions without the formation of oxygen gradients that would occur in a flask-based batch culture.

Niche Differentiation and Environmental Habitat Adaptation In environmental samples, detected microorganisms are often categorized together based on their phylogeny or functional metabolism; however, within these broad categories are a diverse set of species that can each display different characteristics and adaptations. Being able to determine these species-specific adaptations within functional groups of microorganisms allows for a deeper understanding of their ecological role and can explain their co-occurrence in a particular environment. The co-occurrence of organisms that share the same functional role does not follow the ecological competitive exclusion principle, which proposes that two or more species competing for the same resource cannot co-exist in stable populations over long periods of time (176, 177). However, multiple species within the same functional group of microorganisms are often found together in the same environment. This co-occurrence has been investigated with several functional groups of microorganisms, including sulfur-oxidizing bacteria (178, 179), methanotrophic bacteria (180, 181), and AOM (88, 182, 184)." The co-occurrence of multiple AOM found almost ubiquitously in soil (182), freshwater (108) and marine environments (183, 184) suggests that these species have, to some extent, undergone niche differentiation while conserving their overall ammonia-oxidizing metabolism. This niche differentiation is the focus of many recent physiological and genomic studies comparing cultured AOM (88, 107, 185). AOM that do not co-occur can be distinguished from each other based on physiological adaptions to their environmental habitat. Known physiological adaptations are often related to substrate concentration, temperature, or salinity requirements. The adaptation to different substrate concentrations is of particular interest between AOB species

" 19" because they vary widely across species; with the km of N. europaea around 30-61 μM, while the km of N. oligotropha is about ten-fold lower, between 1.9-4.2 μM (80). With microbial genomic sequencing on the rise, these physiological adaptations can now be investigated across species on the genomic level. Being able to connect genomic trends to physiologic adaptations of species to different environmental parameters is the next step in understanding how these adaptations arise.

Microbial Community Interactions Interactions within a microbial community play an important role in understanding how microbial ecosystems function and respond to change. The study of microbial community function has been on the rise since the onset of meta-omic techniques in the early 21st century such as metagenomics (186, 187), metatranscriptomics (188, 189), and metaproteomics (190, 192). These broad reaching methods allow for the potential identification of the total number of genes (DNA), mRNAs, or proteins found in an environmental sample, respectively. While these methods are widely used, often in tandem, to gain a better understanding of the microbial community and its potential in a given environment (193-195), the information gathered if often too broad to detect interactions at the species level. Laboratory-based ecophysiological and biochemical studies performed with axenic cultures represent the other end of the spectrum compared to in situ environmental –omic sampling. These allow for the characterization of species level differences and adaptations but may not be representative of how microorganisms respond in a natural community structure. Well-characterized enrichment cultures and co- cultures of community members from the same environment are often used to mediate the challenges faced by both axenic cultures and environmental samples (196-197). These types of cultures offer manageable numbers of known community members and a comparatively high total microbial biomass, to allow species level interactions to be identified and characterized. These traits allow for proteomic and transcriptomic data to be confidently assigned to community members with known genomic content. This approach has been utilized for many different types of microbial communities (198) including methanotrophic communities (196). It has also been used to investigate bacterial-fungal (199), and more specifically, AOB-NOB interactions (147). The interactions nitrifying microorganisms have with each other and other non-nitrifying community members is important for characterizing nitrifying communities in both natural

" 20" environments and engineered systems like WWTPs. Positive interactions between AOB as well as with NOB (10, 147) and heterotrophic bacteria (7-9, 16) have been reported previously. Interactions between AOB and NOB stem from their mutually beneficial metabolisms. In comparison, the interaction between AOB and heterotrophic bacteria is relatively unclear. Heterotrophic bacteria are known to scavenge soluble microbial products secreted by AOB (200), but how the AOB benefit and why they exhibit a faster growth rate in the presence of heterotrophic bacteria is currently unknown. Further studies investigating AOB-heterotroph interactions will help elucidate the cellular response of AOB to the presence of naturally occurring community members.

Project Goals

+ The first goal of this project was to characterize AOB adapted to low NH4 environments, both at the physiological and genomic levels. Physiological characterization of AOB was carried out through culture-based experiments that measured growth over a range of abiotic factors that

+ - included gradients of pH, oxygen concentration, NH4 concentrations, and NO2 concentrations. + The ability of each AOB species to use urea as a source of NH4 was also tested. In addition, the genomes of Nitrosomonas sp. Is79, Nitrosomonas sp. Is341, Nitrosomonas JL21, N. oligotropha, Nitrosomonas GH22, and Nitrosomonas HPC101 have been sequenced, annotated and compared. Each AOB utilized in this study has at least a draft genome sequence, with three having fully sequenced and closed genomes. I hypothesize that the AOB from Nitrosomonas cluster 6a and cluster 7 will each have conserved genomic traits relating to genes involved in ammonia- oxidation, carbon fixation and nitrogen oxide metabolism. These conserved genomic traits should lend hypothesis to or explanations of the observed ecophysiological adaptations of Nitrosomonas cluster 6a and cluster 7 AOB. The second goal of this project was to determine the effect of bacterial community members, on the proteome of the AOB Nitrosomonas sp. Is79. The bacterial members of the well-characterized enrichment culture G5-7 were used to conduct these experiments. Batch cultures and chemostat continuous culture growth experiments were conducted with both co- cultures and community cultures involving Nitrosomonas sp. Is79. I hypothesize that Nitrosomonas sp. Is79 will interact positively with both NOB and heterotrophic bacteria through different cellular mechanisms. In addition, I hypothesize the interaction with NOB to be based on

" 21" the removal of waste products by the NOB and the subsequent reduction of stress for Nitrosomonas sp. Is79; where the interaction with heterotrophic bacteria is based on the heterotrophic bacteria supplementing Nitrosomonas sp. Is79 with complex organic compounds.

" 22" CHAPTER 1

Physiological and Genomic Comparison of Ammonia-oxidizing Bacteria Adapted to Different Ammonium Concentrations

Christopher Sedlacek, Brian McGowan, Yuichi Suwa, Luis Sayavedra-Soto, Hendrikus J. Laanbroek, Lisa Y. Stein, Jeannette M. Norton, Martin G. Klotz, and Annette Bollmann

" 23" Abstract

Betaproteobacterial ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas phylogenetically cluster together in groups, where species in each group share similar physiological adaptations to abiotic environmental conditions. Here we present a study investigating the physiological adaptations and associated genomic characteristics that differ between Nitrosomonas cluster 6a and Nitrosomonas cluster 7 AOB, which are generally adapted

+ to low and high ammonia concentrations respectively. The influence of ammonium (NH4 ) - concentration, nitrite (NO2 ) concentration, O2 headspace concentration, and pH on the growth rate of AOB from Nitrosomonas cluster 6a (Nitrosomonas sp. Is79, Nitrosomonas sp. AL212, Nitrosomonas oligotropha, Nitrosomonas sp. Is341, Nitrosomonas sp. JL21) and from Nitrosomonas cluster 7 (Nitrosomonas eutropha, Nitrosomonas sp. GH22, Nitrosomonas sp. HPC101) was investigated. The growth rates of Nitrosomonas cluster 6a AOB decreased with

+ - increasing NH4 concentrations above 1 mM and with increasing NO2 concentrations above 0.5 mM. In contrast, the growth rates of Nitrosomonas cluster 7 AOB were not negatively affected

+ - by high NH4 or NO2 concentrations. In silico genomic comparisons were utilized to identify conserved genomic traits that correlated with the observed physiological growth adaptations of Nitrosomonas cluster 6a and Nitrosomonas cluster 7 AOB. The genomes of Nitrosomonas cluster 7 AOB encode for a suite of genes necessary for the production nitric oxide (NO) and nitrous oxide (N2O) as well as multiple terminal oxidases that are absent in Nitrosomonas cluster 6a AOB. In addition, all Nitrosomonas cluster 6a AOB possess two different forms of ribulose- 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for urea utilization. Linking physiological adaptations to conserved genomic traits is essential to understand how eutrophication in the environment may affect AOB community succession and subsequently other processes such as the release of the nitrogenous greenhouse gases NO and

N2O.

" 24" Introduction Nitrification plays a crucial role in nitrogen cycling within and between terrestrial, freshwater, and marine ecosystems . Aerobically, nitrification is completed by chemolithoautotrophic ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB). AOB utilize ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) enzyme complexes to perform the first step of

- nitrification, the oxidation of ammonia (NH3) to nitrite (NO2 ) via hydroxylamine (NH2OH) (26, - - 131). NOB oxidize NO2 to NO3 to complete nitrification (27, 28, 154). All AOB isolated to date belong to monophyletic groups within the Gamma- or Beta- proteobacteria (139, 141). Betaproteobacterial AOB belong to either the Nitrosospira or the Nitrosomonas genus. Species within the genus Nitrosomonas can be subdivided into 6 clusters based on 16S rRNA and amoA sequences (80, 141). AOB in each of the 6 clusters exhibit different physiological adaptations (141). These physiological adaptations variety from growth at sub-zero temperatures (Nitrosomonas cyrotolerans lineage) (202), with high salt requirements (Nitrosomonas cluster 6b, the Nitrosomonas marina lineage and the Nitrosomonas sp. Nm143 lineage), to growth in moderately eutrophic environments (Nitrosomonas cluster 8, the Nitrosomonas communis lineage) (141). Nitrosomonas cluster 6a AOB (the Nitrosomonas oligotropha lineage) have an AMO with a high substrate affinity (Km) for NH3 and their growth + - is sensitive to high NH4 and NO2 concentrations (141). In contrast, Nitrosomonas cluster 7

AOB (the Nitrosomonas europaea/mobilis lineage) have an AMO with a low Km for NH3 and + - their growth is insensitive to high NH4 and NO2 concentrations (141). Nitrosomonas cluster 6a and 7 AOB are often found in oligotrophic and eutrophic freshwater environments, respectively

+ (26, 141), where NH4 , CO2, and O2 concentrations are in constant flux. To compete for + resources such as NH4 , CO2, and O2, which can be limiting in the environment, AOB employ several different adaptive traits. AOB can utilize multiple forms of RuBisCO or terminal oxidases that have different substrate affinities (2, 116-120). Select AOB can also produce carboxysomes in place of multiple forms of RuBisCO (2, 116-120, 203). Nitrification is beneficial in both natural and engineered environments, where it provides oxidized inorganic nitrogen compounds to the surrounding location (1, 131) and is involved in the removal of inorganic nitrogen compounds from wastewater (204, 205). However, in environments supplemented with anthropogenic nitrogen, nitrification contributes to increased

" 25" nutrient runoff (59, 60), eutrophication of downstream aquatic ecosystems (63-65), and the release of nitrogenous greenhouse gases (GHGs) such as nitric oxide (NO) or nitrous oxide

(N2O) (70, 206). Although all AOB in microbial communities fulfill the same functional role, i.e. ammonia oxidation, understanding subtle species-level characteristics is essential in determining the ecological impact of individual AOB species (2, 70, 116-120, 141). Identifying and linking conserved genomic traits and physiological adaptations will allow for better modeling of the functional potential of AOB species across a wide range of ecosystems. The availability of whole genome sequencing has made it possible to identify conserved genomic traits within and between groups of microorganisms that share similar physiological adaptations. This approach has been utilized to investigate Saccharomyces cerevisiae strains that are adapted to low temperatures (207), Prochlorococcus ecotypes adapted to different light intensities (208), and the ability of select Bacillus species to grow in alkaline environments (3). To date, AOB genomic comparisons have been limited by the small number of available AOB genomes and the phylogenetic distance between the AOB that have been sequenced (134). Investigating genomic traits conserved within but not between Nitrosomonas cluster 6a and 7 AOB will shed light on observed physiological adaptations specific to AOB within each cluster (116-120). This study physiologically and genomically compares 5 Nitrosomonas cluster 6a AOB, Nitrosomonas sp. Is79 (2), Nitrosomonas sp. AL212 (142, 120), Nitrosomonas oligotropha (140), Nitrosomonas sp. JL21 (209) and Nitrosomonas sp. Is341 as well as 3 Nitrosomonas cluster 7 AOB Nitrosomonas eutropha (119, 140, 210), Nitrosomonas sp. HPC101 (142) and Nitrosomonas sp. GH22 (209). This is the first report of a comparison between multiple AOB genomes from more than one Nitrosomonas cluster.

" 26" Material and Methods Cultures and media Cultures: Nitrosomonas sp. Is79 (2), N. eutropha (119, 140, 210), Nitrosomonas sp. AL212 (120, 142), N. oligotropha (140) Nitrosomonas sp. JL21 (209), Nitrosomonas sp. Is341 (This study), Nitrosomonas sp. HPC101 (142) and Nitrosomonas sp. GH22 (209) were utilized (Table 1).

Medium to cultivate AOB: A mineral salts (MS) medium containing 10 mM NaCl, 1 mM KCl, 1

-1 mM CaCl2!2H2O, 0.2 mM MgSO4!7H2O and 1 mL liter trace elements solution was used for all growth experiments (169, 211). HEPES buffer was added in a 4-fold molar ratio to the initial

+ NH4 concentration and the pH was adjusted to 7.8 before autoclaving (169). Sterile KH2PO4 was added to a final concentration of 0.4 mM after autoclaving.

Growth experiments

+ AOB growth rates were determined across a range of initial medium pH (6 - 9), NH4 (0.1 – 10 - mM), NO2 (0.1 – 5 mM), and headspace O2 concentrations (0% - 21.5%). The initial pH was adjusted with 1 M NaOH before autoclaving. O2 headspace concentrations were attained by equilibrating MS medium in serum bottles under anaerobic conditions overnight in an anaerobic chamber (Coy laboratory Products). After equilibration, the bottles were sealed with rubber stoppers and different O2 concentrations were achieved by exchanging volumes of the anaerobic headspace with sterile filtered air through a syringe filter (0.2 µm). The ability of AOB to utilize urea as a source of NH3 was investigated through the addition of sterile filtered (0.2 µm) urea + (0.25 mM) to autoclaved MS medium containing NH4 (1 mM). Samples from all experiments were taken at regular intervals and centrifuged (20 min, 28,000 x g). Cell free supernatants were

o + - stored at -20 C for chemical analysis of NH4 and NO2 concentrations. Growth rates were - determined by calculating the slope of the log transformed NO2 concentrations against time, assuming a correlation with growth (169, 212).

+ - Chemical analysis: Colorimetric assays were used to determine NH4 and NO2 concentrations in cell free supernatants (169, 213, 214).

" 27" Table 1. AOB isolation environment and Nitrosomonas cluster affiliation.

General AOB Site of Physiological Species / Isolate Reference Isolation Traits Name Lake Sediment Nitrosomonas sp. Is79 Lake Drontermeer Bollmann et al., 2013 The Netherlands Sensitive to high Soil + - N. oligotropha Koops et al., 1991 NH4 and NO2 Hamburg, Germany Nitrosomonas concentrations Activated sludge Nitrosomonas sp. AL212 Suwa et al., 2011 cluster 6a Tokyo, Japan High affinity (K ) Activated sludge m Nitrosomonas sp. JL21 Suwa et al., 1994 for NH3 Tokyo, Japan Lake sediment Nitrosomonas sp. Is341 Lake Drontermeer This Study The Netherlands Insensitive to high Sewage Plant + - N. eutropha Watson and Mandel, 1971 NH4 and NO2 Chicago, USA Nitrosomonas concentrations Activated sludge Nitrosomonas sp. HPC101 Suwa et al., 1997 cluster 7 Tokyo, Japan Low affinity (K ) Activated sludge m Nitrosomonas sp. GH22 Suwa et al., 1994 for NH3 Tokyo, Japan

" 28" Growth for whole genome DNA isolation N. oligotropha, Nitrosomonas sp. JL21, Nitrosomonas sp. Is341, Nitrosomonas sp. HPC101 and Nitrosomonas sp. GH22 were each grown to late log phase as 1 L batch cultures (minimum 6 L)

+ in MS medium with 10 mM NH4 . Cell biomass was collected by centrifugation (20 min, 22,000 x g, 4oC), pooled at stored at -80oC for whole genome DNA isolation.

Whole genome DNA isolation N. oligotropha, Nitrosomonas sp. JL21, Nitrosomonas sp. Is341, Nitrosomonas sp. HPC101 and Nitrosomonas sp. GH22 DNA was isolated with the Joint Genome Institute (JGI) bacterial genomic DNA isolation using cetyltrimethylammonium bromide (CTAB) protocol (http://my.jgi.doe.gov/general/protocols/JGI-Bacterial-DNA-isolation-CTAB-Protocol-2012.pdf). DNA was stored at -80oC until whole genome sequencing.

Next generation sequencing N. oligotropha, Nitrosomonas sp. JL21, Nitrosomonas sp. Is341, Nitrosomonas sp. HPC101 and Nitrosomonas sp. GH22 libraries were prepped with the Nextera XT library prep kit as per manufactures instructions (Illumina, Madison, WI). The libraries were sequenced on an Illumina 1.9 MiSeq instrument at the Center for Genome Research and Biocomputing (CGRB) at Oregon State University. Libraries of N. oligotropha, Nitrosomonas sp. JL21, Nitrosomonas sp. Is341, Nitrosomonas sp. HPC101 and Nitrosomonas sp. GH22 generated paired end reads of 17,153,696, 14,546,069, 17,182,123, 8,383,203 and 9,187,862 respectively, which were assembled into 70, 87, 296, 32 and 80 contigs, and resulted in overall coverage of 1287, 933, 927, 1682 and 2060 respectively.

Genome assembly and annotation De novo assembly of N. oligotropha, Nitrosomonas sp. JL21, Nitrosomonas sp. Is341, Nitrosomonas sp. HPC101 and Nitrosomonas sp. GH22 was performed using Genomics Workbench 7.5 with the Bacterial Genome Finishing Module (CLC bio, Boston, MA), run on a workstation with an AMD Opteron 2.10 GHz 16-core processor with 128 GB DDR3 ECC random access memory (RAM), with standard default settings. The genomes were annotated with Prokka version 1.10 on a quadcore i7 workstation with 32 GB DDR3 running Ubuntu 14.04

" 29" long-term support (LTS) (215). Annotations were first called from a specialized library comprised of N. europaea, N. eutropha, Nitrosomonas sp. Is79 and Nitrosomonas sp. AL212, then from the general Prokka database. Genome comparisons were performed using Genomics Workbench 7.5 with a database assembled from the CDS translations from N. eutropha, N. oligotropha, Nitrosomonas sp. Is79, Nitrosomonas sp. AL212, Nitrosomonas sp. JL21, Nitrosomonas sp. Is341, Nitrosomonas sp. HPC101 and Nitrosomonas sp. GH22

16S rRNA sequence analysis All 16S rRNA sequences were edited with 4Peaks (A. Griekspoor and T. Groothuis, The Netherlands Cancer Institute). The sequences were aligned using ARB (216). Phylogenetic trees were constructed using the neighbor-joining algorithm in ARB with Jukes-Cantor correction.

" 30" Results and Discussion Phylogenetic and Physiologic Comparison All 8 AOB utilized in this study are members of either Nitrosomonas cluster 6a or Nitrosomonas cluster 7 within the genus Nitrosomonas in the Betaproteobacteria subphylum

+ (Fig. 3). Nitrosomonas cluster 6a AOB exhibited their highest growth rates when the initial NH4 + concentration was ≤ 1 mM. Growth rates declined as NH4 concentration increased above this threshold level (Fig. 4A,B). In contrast, the growth rates of Nitrosomonas cluster 7 AOB

+ increased as the initial NH4 concentration increased up to 5 mM and did not decrease at higher + NH4 concentrations (Fig. 4A,B). In addition, the growth rates of Nitrosomonas cluster 6a AOB - but not Nitrosomonas cluster 7 AOB decreased with increasing initial NO2 concentrations (Fig. - 4C). No Nitrosomonas cluster 6a AOB grew in the presence of 5 mM NO2 (Fig. 4C). The sensitivity of Nitrosomonas cluster 6a AOB but not Nitrosomonas cluster 7 AOB to high

+ - concentrations of NH4 and NO2 is in line with previous observations (141). These results suggest a conserved sensitivity of Nitrosomonas cluster 6a AOB but not Nitrosomonas cluster 7 AOB to substrate inhibition and toxic waste product accumulation. All AOB tested, exhibited decreased growth rates as O2 headspace concentration decreased (Fig. 4D) and exhibited their highest growth rates at neutral pH values (7-8) (Fig. 4E), which confirmed earlier observations (88, 141). Together, the physiological adaptations of Nitrosomonas cluster 6a and 7 AOB explain their observed presence and abundance in different natural and engineered environments (143, 164, 184, 217-219). In agreement with past reports (140, 141, 209), most Nitrosomonas cluster 6a AOB can utilize urea as a source of NH3, however, no Nitrosomonas cluster 7 AOB characterized to date can utilize this substrate (Fig. 4F). The ability to utilize urea as an alternative energy source

+ should be an advantageous adaptation for AOB in environments with low NH4 concentrations. However, this capability is not conserved among all Nitrosomonas cluster 6a AOB (Fig. 4F) (140, 141, 209), moreover, the ability to utilize urea as an alternative energy source has been observed in AOB species from Nitrosomonas cluster 8, the Nitrosomonas communis lineage, which are adapted to moderately eutrophic environments (141). Together, this suggests that urea utilization in Nitrosomonas AOB species is a species-specific physiological adaptation and may

+ not be directly related to an adaptation to low NH4 concentrations.

" 31"

Figure 3. Neighbor-joining tree of AOB based on 16S rRNA gene nucleotide sequences. Select phylogenetic clusters with the genus Nitrosomonas are noted. Bootstrap values > 50 of 100 replicates are shown at the nodes. Species utilized in this study are bolded.

" 32"

53 Nitrosomonas oligotropha Nitrosomonas sp. Nm47 Nitrosomonas sp. Is341 Nitrosomonas sp. Nm86 Nitrosomonas 98 Nitrosomonas sp. Is79 100 Nitrosomonas ureae cluster 6a 100 Nitrosomonas sp. AL212 100 Nitrosomonas sp. Nm59 98 Nitrosomonas sp. JL21 ] 100 Nitrosomonas aestuarii 74 Nitrosomonas marina 100 Nitrosomonas eutropha 98 Nitrosomonas sp. GH22 100 Nitrosomonas sp. HPC101 Nitrosomonas 94 Nitrosomonas europaea cluster 7 98 Nitrosomonas halophila ] Nitrosococcus mobilis 100 Nitrosomonas communis 100 Nitrosomonas nitrosa 90 Nitrosospira sp. NpAV 56 Nitrosovibrio tenuis 100 Nitrosospira briensis 70 Nitrosospira multiformis Gallionella ferruginea 100 Methylophilus leisingeri 94 Azoarcus tolulyticus Leptothrix mobilis Alcaligenes faecalis Escherichia coli K12

0.10

" 33"

+ - Figure 4. The influence of: a,b) initial NH4 concentration, c) initial NO2 concentration, d) initial culture pH and e) initial headspace O2 concentration on the growth rate of AOB. f) The ability of AOB to utilize urea as a source of NH3. Nitrosomonas cluster 6a AOB symbols are closed with black lines: N. oligotropha (!), Nitrosomonas sp. Is79 ("), Nitrosomonas sp. Is341 (#), Nitrosomonas sp. JL21 ("), Nitrosomonas sp. AL212 (#) and Nitrosomonas cluster 7 AOB symbols are open with grey lines: N. eutropha ($), Nitrosomonas sp. GH22 (%), Nitrosomonas sp. HPC101 (!).

" 34"

0.07 A B 0.07

0.06 0.06

0.05 0.05 ) ) -1 -1

0.04 0.04

0.03 0.03 Growth RateGrowth (h Growth RateGrowth (h 0.02 0.02

0.01 0.01

0 0 0 2 4 6 8 10 0 0.2 0.4 0.6 0.8 1 Initial Ammonium Concentration [mM] Initial Ammonium Concentration [mM]

C 0.04 D

0.035 0.04

0.03 ) ) -1 0.03

0.025 -1

0.02 0.02 0.015 Growth RateGrowth (h Growth RateGrowth (h

0.01 0.01

0.005

0 0 0 1 2 3 4 5 6 6.4 6.8 7.2 7.6 8 8.4 8.8 9.2 Initial Nitrite Concentration [mM] Initial pH

E 0.035 F Ability to use Urea as an Ammonium Source 0.03 N. oligotropha + 0.025

) Nitrosomonas sp. Is79 - -1 0.02 Nitrosomonas sp. Is341 + 0.015 Nitrosomonas sp. JL21 + Growth RateGrowth (h 0.01 Nitrosomonas sp. AL212 +

0.005 N. eutropha -

0 Nitrosomonas sp. HG22 - 0 5 10 15 20 25 Initial Oxygen Concentration (%) Nitrosomonas sp. HPC101 -

" 35" Genomic comparison General genome characteristics All Betaproteobacterial AOB genomes sequenced to date are between 2-4 Mbps (Table 2) (2, 116, 118-120). This is relatively small compared to other sequenced Betaproteobacterial genomes, which are often larger than 5 Mbps (134). Nitrosomonas cluster 6a AOB have larger genomes (~10-30% larger) than Nitrosomonas cluster 7 AOB (Table 2) (2, 116, 119, 120), which was unexpected because of their adaptation to low substrate concentrations and therefore low energy environments (Fig. 4A,B). All of the AOB genomes presented here possess a single rrn operon and at least 38 tRNAs that encode all 20 essential amino acids, with the exception of a few genes that were not detected in the draft genome sequences of Nitrosomonas sp. Is341, Nitrosomonas sp. JL21 and Nitrosomonas sp. HPC101 (Table 3). Interestingly, all the genomes of Nitrosomonas cluster 6a AOB lack 2 tRNAs, one for glycine (CCC) and one for proline (CGG), that are found in all 3 of the genomes of Nitrosomonas cluster 7 AOB (Table 3). The difference in encoded tRNA genes may be due to different codon usage preferences between Nitrosomonas cluster 6a and 7 AOB. All Nitrosomonas cluster 6a AOB encode at least one other tRNA gene for both glycine and proline. How these differences in tRNA inventory effect the growth of Nirosomonas cluster 6a and 7 AOB is currently unknown. In addition Nitrosomonas sp. HPC101 is the only AOB to date to encode a pyrolysine (pylT) tRNA (Table 3). For pyrolysine to be incorporated into proteins an aminoacyl- tRNAsynthetase (pylS) is needed to charge the pylT derived tRNA (220, 221). The draft genome sequence of Nitrosomonas sp. HPC101 does not encode a plyS gene, but a confirmatory PCR is needed to verify its presence or absence in the genome of Nitrosomonas sp. HPC101. To date, methyltransferases involved in methanogenesis are the only proteins characterized to contain pyrolysine (220, 222, 223). The draft genome sequence of Nitrosomonas sp. HPC101 does not appear to encode any related methyltransferases but confirmatory PCRs are needed to verify their presence or absence in the Nitrosomonas sp. HPC101 genome. Further investigation of the Nitrosomonas sp. HPC101 draft genome sequence will provide insights into any possible pyrolysine containing proteins. Proteins containing pyrolysine are often initially annotated as two separate proteins separated by an amber stop codon (UAG). If detected, any pyrolysine containing proteins, may shed light on why Nitrosomonas sp. HPC101 is the only AOB to date known to encode the 22nd amino acid.

36" " Table 2. General Characteristics of Nitrosomonas cluster 6a and 7 AOB genomes.

Nitrosomonas cluster 6a Nitrosomonas cluster 7 N. oligotropha Is791 Is341 JL21 AL2122 N. eutropha3 GH22 HPC101 Genome sequence* DS CGS DS DS CGS CGS DS DS Contigs (#) 70 1 296 87 1 1 80 32 Size (Mbp) 3.12 3.78 3.55 3.17 3.34 2.78 2.54 2.52 G+C content (%) 49.2 45.4 46.9 47.8 44.8 48.5 48.1 49.3 Total genes 2947 3597 3458 2961 3238 2775 2445 2386 Protein coding genes 2908 3553 3419 2922 3194 2699 2405 2344 Coding Region (%) 98.7 98.8 98.9 98.7 98.6 97.3 98.4 98.2 rRNA operons 1 1 1 - 1 1 1 1 tRNA genes 38 38 39 38 38 41 39 41 *DS=Draft genome sequence, CGS=Closed genome sequence - = Not detected Is79= Nitrosomonas sp. Is79, Is341= Nitrosomonas sp. Is341, JL21= Nitrosomonas sp. JL21, AL212= Nitrosomonas sp. AL212, GH22= Nitrosomonas sp. GH22 and HPC101= Nitrosomonas sp. HPC101. 1) Bollmann et al., 2013 2) Suwa et al., 2011 3) Stein et al., 2007

37# # Table 3. tRNA genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes. Nitrosomonas Cluster 6a Nitrosomonas Cluster 7 N. oligotropha Is791 Is341 JL21 AL2122 N. eutropha3 GH22 HPC101 tRNAs Ala (TGC) + + ++ + + + + + Ala (GGC) + + + + + + + + Arg (CCT) + + + + + + + + Arg (ACG) + + + + + + + + Arg (CCG) + + + + + + + + Arg (TCT) + + + + + + + + Asn (GTT) + + - + + + + + Asp (GTC) + + + + + + + + Cys (GCA) + + + + + + + + Gln (TTG) + + + + + + + + Glu (TTC) + + + + + + + + Gly (TCC) + + + + + + + + Gly (GCC) + + + + + + + + Gly (CCC) - - - - - + + + His (GTG) + + + + + + + + Ile (GAT) + + ++ + + + + + Leu (TAA) + + + + + + + + Leu (TAG) + + + + + + + + Leu (CAG) + + + + + + + + Leu (CAA) + + + + + + + + Leu (GAG) + + + + + + + + Lys (TTT) + + + + + + + + Met (CAT) +++ +++ - +++ +++ +++ +++ +++ Phe (GAA) + + + + + + + + Pro (GGG) + + ++ + + + + + Pro (TGG) + + + + + + + + Pro (CGG) - - - - - + + + Pyl (CTA) ------+ Ser (TGA) + + + + + + + + Ser (GCT) + + + + + + - + Ser (CGA) + + + + + + - + Ser (GGA) + + + + + + + + Thr (GGT) + + + + + + + + Thr (CGT) + + + + + + + + Thr (TGT) + + ++ + + + + + Trp (CCA) + + + + + + + - Tyr (GTA) + + + + + + + + Val (TAC) + + + + + + - + Val (GAC) + + ++ + + + + + Val (CAC) - - + - - + + + * Each gene copy is represented with a (+) Is79= Nitrosomonas sp. Is79, Is341= Nitrosomonas sp. Is341, JL21= Nitrosomonas sp. JL21, AL212= Nitrosomonas sp. AL212, GH22= Nitrosomonas sp. GH22 and HPC101= Nitrosomonas sp. HPC101. - = Not detected 1) Bollmann et al., 2013 2) Suwa et al., 2011 3) Stein et al., 2007

38# # Energy generation Ammonia oxidation All previously sequenced Betaproteobacterial AOB genomes encode for 2 or 3 nearly identical copies of the amo (amoCAB or amoCAB-orf4-orf5) and hao (hao-orf2-c554-cm552) gene clusters (2, 116, 118-120). The sequence similarity of these large repeated regions is problematic in the assembly of whole genome sequences. All the AOB reported here encode most of the essential genes for ammonia oxidation (Table 4). However, all 5 draft genome sequences reported here lack at least one gene from either the amo or hao gene cluster (Table 4). These missing genes are likely present in the genomes of these 5 AOB, as they all have the ability to chemolithoautotrophically grow with NH3 as a sole energy source (Fig. 4). It is also presumed that all 5 of these draft genome sequences encode at least 2 and possibly 3 copies of both the amo and hao gene clusters, based on previously fully sequenced AOB genomes (2, 116, 118-120). To resolve the number of repeated amo and hao gene clusters encoded in each AOB genome, a combination of whole genomic DNA restriction enzymatic digestion and southern blotting can be used (224). Briefly, whole genomic DNA from each of the 5 AOB with draft genome sequences would be isolated, digested with a restriction enzyme, size fractionated on an agarose gel and blotted onto a nylon membrane. Fluorescent or radio labeled DNA probes for amo and hao genes could then be used to determine the number of copies of these genes present in each genome (224). Minimally, probes for amoC, amoA, orf4, orf5, hao and cytochrome cm552 would need to be utilized to ensure the correct number of operon and singleton gene copies are identified. To date, the red copper protein, nitrosocyanin, has only been found in the genomes of AOB (116, 117, 119, 120). A biochemical study of nitrosocyanin revealed that it may have a catalytic role in ammonia oxidation, nitric oxide reduction or as an electron transfer cytochrome oxidase like protein (225) This has prompted a hypothesis that nitrosocyanin likely is in some way essential to obligate NH3 lithotrophy in AOB (117). However, nitrosocyanin is absent in the genome of 3 Nitrosomonas cluster 6a AOB reported here, including the closed Nitrosomonas sp. Is79 genome (Table 4). This finding seems to refute the hypothesis that nitrosocyanin is essential to obligate NH3 lithotrophy in AOB, but it is possible that a functionally redundant enzyme is

39# #

Table 4. Ammonia oxidation related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes. Nitrosomonas Cluster 6a Nitrosomonas Cluster 7 N. N. Is791 Is341 JL21 AL2122 GH22 HPC101 oligotropha eutropha3 Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: ABD07 Nit79A3 ABF85 ABF87 NAL212 Neut ABF86 ABO04 Ammonia Monooxygenase Gene Clusters #1) amoC/A/B 2816/17/- 471/72/73 1012/11/- -/1063/64 797/98/99 2078/77/76 1662/-/- 2202/03/04 orf4/orf5 -/1124 474/75 -/- 2826/ 2827 800/01 2075/74 -/- 2205/ 06 copC/copD -/- -/- -/- -/- 802/03 -/- -/- -/-

#2) amoC/A/B -/-/- 1079/80/81 -/3003/04 -/-/- 1386/87/88 2319/18/17 -/-/- -/-/- orf4/orf5 -/- -/- -/- -/- -/- 2316/15 2280/ 2281 -/- copC/copD -/- -/- -/- -/- -/- 2314/13 -/- -/-

#3) amoC/A/B -/-/- 2886/85/84 2736/-/- -/-/- 2606/05/04 -/-/- -/-/- -/-/- orf4/orf5 -/- 2883/82 -/ - -/- 2603/02 -/- -/- -/-

1233/ 2303/ 2258/ amoC singletons - 2736 - 1593 1127 1595 2818 2360 copC/D 1116/1117 3552/1551 1829/1145 2350/1571 - - 293/294 1881/1881

Nitrosocyanin - - 23 - 897 143 17 19 Ammonium Transporter 322 2491 746 2925 965 - - - amtB Hydroxylamine Oxidoreductase Gene Clusters 1438-39/ 807/ 1280/ 2881/ 1807/ 1672 2336/ 2338/ #1) hao/orf2/c554/c 552 m 40/41/42 08/09/10 79/78/77 80/79/- 06/05/04 /71/70/- 37/38/- 37/36/-

822/ 2138/ 1793/ #2) hao/orf2/c554/c 552 -/-/-/- -/-/-/- -/-/-/- -/-/-/- -/-/-/- m 23/24/25 37/36/35 92/91/90

2942/41/40 2750/49/48 2335/ #3) hao/orf2/c554/c 552 -/-/-/- -/-/-/- -/-/-/- -/-/-/- -/-/-/- m /39 /47 34/33/32 Is79= Nitrosomonas sp. Is79, Is341= Nitrosomonas sp. Is341, JL21= Nitrosomonas sp. JL21, AL212= Nitrosomonas sp. AL212, GH22= Nitrosomonas sp. GH22 and HPC101= Nitrosomonas sp. HPC101. - = Not detected 1) Bollmann et al., 2013, 2) Suwa et al., 2011 3) Stein et al., 2007

40# # being utilized in nitrosocyanin negative AOB. Identifying such an enzyme is complicated by the fact that the exact function of nitrosocyanin in AOB is still unknown.

Terminal oxidases In aerobic bacteria, terminal oxidases perform the last step in the electron transport chain

(227, 228). They are responsible for the transfer of electrons to the final electron acceptor (O2), and in doing so reduce O2 to H2O (227, 228). Bacteria often encode multiple terminal oxidases, because different terminal oxidase complexes can have varying affinities for O2 (228, 229). The genomes of all Nitrosomonas cluster 6a and 7 AOB in this study encode the low affinity cytochrome c oxidase aa3 gene cluster (subunits I-IV) (Table 5). In addition, 2 of the 3 Nitrosomonas cluster 7 AOB genomes also encode 2 other terminal oxidases, the low affinity quinol oxidase bo3 (subunits I-IV) and the high affinity cytochrome c oxidase cbb3 (subunits I- IV-senC) (Table 5). The genome of 3rd Nitrosomonas cluster 7 AOB, Nitrosomonas sp. HPC101, does not appear to encode these additional 2 terminal oxidases, but confirmatory PCRs are needed to determine if these are truly absent in the genome of Nitrosomonas sp. HPC101. If they are present in the genome of Nitrosomonas sp. HPC101, encoding all 3 terminal oxidases may be a conserved trait in Nitrosomonas cluster 7 AOB. Interestingly, none of the Nitrosomonas cluster 7 AOB encode the fixGHIS gene cluster, which has been previously shown to be required for cytochrome c oxidase cbb3 assembly (226). Bacteria from environments with fluctuating O2 levels such as soil or aquatic environments often encode multiple terminal oxidases to adapt to the varying O2 concentrations (227). Aerobic ammonia-oxidation is dependent on the ability of terminal oxidases to capture

O2. Encoding multiple terminal oxidases, including the high affinity cytochrome c oxidase cbb3, may provide Nitrosomonas cluster 7 AOB with a competitive advantage over Nitrosomonas cluster 6a AOB under O2 limited conditions. Pseudomonas putida has been shown to differentially regulate multiple terminal oxidases to adapt to different O2 concentrations (229). Although Nitrosomonas cluster 7 AOB did not appear to have a competitive growth advantage in the low O2 experiments presented here (Fig. 4E), more stringent O2 concentration control is likely necessary to detect potential growth advantages. The investigation of an adaptation to microaerophilic environments requires a tightly controlled system where O2 is supplied as the growth limiting substrate.

41# # Table 5. Terminal oxidase genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes.

Nitrosomonas Cluster 6a Nitrosomonas Cluster 7 N. N. Is791 Is341 JL21 AL2122 GH22 HPC101 oligotropha eutropha3 Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: ABD07 Nit79A3 ABF85 ABF87 NAL212 Neut ABF86 ABO04 Terminal Oxidases Cytochrome c oxidase aa 3 + + + + + + + + (Low-affinity O2 Reductase) Subunit I (COG 843) 1795 385 1108 2711 524 2394 2125 541 Subunit II (COG 1622) 1796 384 1107 2710 523 2395 2124 542 Subunit III (COG 1845) 1792 389 1112 2714 528 2392 2128 538 Subunit IV (COG 3245/3175) 1793 387 1110 2713 526 2393 2127 539

Quinol oxidase bo (Low- 3 - - - - - + + - affinity O2 Reductase) Subunit I (COG 843) - - - - - 696 673 - Subunit II (COG 1622) - - - - - 697 674 - Subunit III (COG 1845) - - - - - 695 672 - Subunit IV (COG 3125) - - - - - 694 671 -

Cytochrome c oxidase cbb3 (High-affinity O2 - - - - - + + - Reductase) Subunit I (fixN/ccoN) - - - - - 1582 1191 - Subunit II (fixO/ccoO) - - - - - 1583 1192 - Subunit III(fixP/ccoP) - - - - - 1584 1193 - Subunit IV (fixQ/ccoQ) ------senC - - - - - 1585 1194 - Is79= Nitrosomonas sp. Is79, Is341= Nitrosomonas sp. Is341, JL21= Nitrosomonas sp. JL21, AL212= Nitrosomonas sp. AL212, GH22= Nitrosomonas sp. GH22 and HPC101= Nitrosomonas sp. HPC101. - = Not detected 1) Bollmann et al., 2013 2) Suwa et al., 2011 3) Stein et al., 2007

42# # Ammonia transporter The ammonia monooxygenase enzyme complex, which resides in the cytoplasmic membrane, is responsible for the first step in ammonia oxidation (114). Based on its similarity to the particulate methane monooxygenase enzyme, its active site is predicted to be on the periplasmic side of the membrane (72, 105, 228). Based on this generally accepted model of the ammonia monooxygenase machinery, AOB do not require active ammonia transporters to acquire NH3 for ammonia oxidation. However, the genomes of all of the Nitrosomonas cluster 6a AOB encode an amtB type ammonia transporter, which is not encoded in any of the Nitrosomonas cluster 7 AOB genomes presented here (Table 4). The conserved presence of this

+ transporter in AOB adapted to low NH4 environments is curious because AOB use NH3 for energy generation and assimilation. Actively transporting NH3 into the cytoplasm appears to be counterproductive for energy generation in the current model. It of interest to note that the

+ genome of Nitrosomonas europaea, which is adapted to high NH4 environments also encoded an amtB type ammonia transporter (116). The direct impact of the NH3 transporter on the growth + of Nitrosomonas cluster 6a AOB at different NH4 concentrations and whether it is involved in capturing NH3 for catabolism, assimilation or both is currently unknown.

Alternative Energy Sources Urea Utilization

The ability of AOB to intracellularly hydrolyze urea into NH3 and CO2 is advantageous, as it provides both substrate for energy generation and carbon for cellular growth (225-227). However, this trait is not universally conserved among all AOB (140, 141, 209). All of the Nitrosomonas cluster 6a AOB, except Nitrosomonas sp. Is79, were able to utilize urea as a source of NH3 for growth (Fig. 4F). In contrast, none of the 3 Nitrosomonas cluster 7 AOB were able to utilize urea as a source of NH3 for growth (Fig. 4F). The genome of each AOB that was able to utilize urea encoded a similar gene cluster for the essential structural and accessory proteins of a urease enzyme (urea amidohydrolase) (ureABCDEFG), along with a urea transporter (Fig. 4F, Table 6). This correlation suggests that AOB able to utilize urea as an energy source, do so through an intracellular urease enzyme. Interestingly, the genomes of all 8 AOB in this study, encode urea carboxylase and two accessory proteins, which make up subunit 1 of an urea amidolyase enzyme (UAL) (Table 6).

43# # Table 6. Urea and hydrogen utilization related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes. Nitrosomonas Cluster 6a Nitrosomonas Cluster 7 N. N. Is791 Is341 JL21 AL2122 GH22 HPC101 oligotropha eutropha3 Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: ABD07 Nit79A3 ABF85 ABF87 NAL212 Neut ABF86 ABO04 Urea Utilization Urease operon + - + + + - - - ureD 514 - 796 567 172 - - - ureA 513 - 795 566 1720 - - - ureB 512 - 794 565 1719 - - - ureC 511 - 793 564 1718 - - - ureE 510 - 792 563 1717 - - - ureF 509 - 791 562 1716 - - - ureG 508 - 790 561 1715 - - - Urea transpoter utp 507 - 789 560 1714 - - -

Urea amidolyase + + + + + - - - Urea carboxylase 979 666 1265 1902 208 2470 2088 1447 Associated protein 1 980 667 1264 1903 209 2474 2091 2357 Associated protein 2 981 668 1263 1904 210 2473 2092 2356 Urea allophanate hydrolyase 908 665 1266 1901 207 - - -

Urea Permease 982 669 1262 1905 211 2472 2089 2358 Hydrogenase Hydrogenase + + - + - - - - hoxF 2714 1351 - 472 - - - - hoxu 2713 1352 - 471 - - - - hoxy 2704 2064 - 470 - - - - hoxh 2703 2065 - 469 - - - - hoxw 897 2189 - 467-468 - - - - hypA 896 2191 - 466 - - - - hypB 895 2192 - 465 - - - - hypF 894 2193 - 464 - - - - hypC 1386 3183 - 2646 - - - - hypD 1387 3184 - 2647 - - - - hypE 1388 3185 - 2648 - - - - Is79= Nitrosomonas sp. Is79, Is341= Nitrosomonas sp. Is341, JL21= Nitrosomonas sp. JL21, AL212= Nitrosomonas sp. AL212, GH22= Nitrosomonas sp. GH22 and HPC101= Nitrosomonas sp. HPC101. - = Not detected 1) Bollmann et al., 2013 2) Suwa et al., 2011 3) Stein et al., 2007

44" " The genomes of all the Nitrosomonas cluster 6a AOB also encode for, allophanate hydrolase, subunit 2 of UAL (Table 6). The presence of one or both of the UAL subunits in the genomes reported here does not correlate with the ability of that AOB to utilize urea (Fig 4; Table 6). This implies that a complete UAL complex in AOB does not bestow the ability to utilize urea (Fig. 4F; Table 6), even though it has been shown to be essential for urea utilization in other microorganisms (225, 226). Urea utilization is a conserved physiological trait among Nitrosomonas cluster 6a AOB,

+ which are adapted to low NH4 concentrations, with a few exceptions (Fig. 4) (140, 141, 209). + Nitrosomonas cluster 6a AOB have a high affinity for NH3 and the ability to create low NH4 + concentrations from the enzymatic degradation of urea could make habitats with low free NH4 concentrations more suitable for these AOB. In contrast, Nitrosomonas cluster 7 AOB have a

+ low affinity for NH3 and are adapted to environments with high free NH4 concentrations. + Therefore, being able to liberate low amounts of NH4 from urea may not be a beneficial adaptation for Nitrosomonas cluster 7 AOB. In line with previous observations, no Nitrosomonas cluster 7 AOB characterized to date have the ability to utilize urea as an energy source (Fig. 4) (140, 141, 209).

H2 oxidation (Hydrogenase) Aerobic bacteria can utilize hydrogen dehydrogenases or hydrogenase enzyme complexes to oxidize H2 under hypoxic conditions (234-236). This process can be directly linked to the reduction of NAD (235). The genomes of Nitrosomonas sp. Is79 (2), N. oligotropha and Nitrosomonas sp. JL21 all encode the genes for a putative [NiFe] hydrogenase enzyme complex, resembling the one encoded for in the genome of Nitrosopira multiformis (Table 6) {Norton:2008bb}. However, in the genome of N. multiformis, the genes for the putative [NiFe] hydrogenase enzyme complex are all found as a single gene cluster (118). In the genome of Nitrosomonas sp. Is79 (2), N. oligotropha and Nitrosomonas sp. JL21 the putative [NiFe] hydrogenase genes are scattered throughout the genome in several different smaller clusters (Table 6). The functionality and regulation of the hydrogenase in N. multiformis has not yet been investigated, therefore it is unknown how splitting the single gene cluster into multiple gene clusters affects the functionality of the putative hydrogenases in Nitrosomonas sp. Is79 (2), N. oligotropha or Nitrosomonas sp. JL21. Overall, it appears that not all Nitrosomonas cluster 6a

45# # AOB have the ability to utilize H2 as the genomes of Nitrosomonas sp. AL212 and Nitrosomonas sp. Is341 do not encode any putative [NiFe] hydrogenase genes (Table 6). Assuming the predicted [NiFe] hydrogenases in the AOB mentioned above are functional, it has been hypothesized that they may reduce the amount of reverse electron flow AOB perform (118). Increasing the amount of NADH produced through a hydrogenase enzyme complex and decreasing the amount generated through reverse electron flow would allow AOB to acquire more energy from ammonia-oxidation. The genomes of the 3 Nitrosomonas cluster 7 AOB investigated did not encode any of the putative [NiFe] hydrogenase genes found in the genomes of several of the Nitrosomonas cluster 6a AOB (Table 6). However, both N. eutropha and another Nitrosomonas cluster 7 AOB, N. europaea, have been shown to grow anaerobically with hydrogen as an electron donor {Anonymous:taLT78NF}. This type of grow requires a hydrogenase, but to date, no hydrogenase genes have been identified in either of the closed genomes of N. eutropha and N. europaea (116, 118). Whether or not other Nitrosomonas cluster 7 AOB such as Nitrosomonas sp. GH22 or Nitrosomonas sp. HPC101 have the ability to anaerobically grow on hydrogen is currently unknown, as direct physiological experiments have not been conducted. In silico analysis is not possible because the genes encoding the suspected hydrogenases in N. eutropha and N. europaea have not been identified. It appears that AOB from Nitrosomonas cluster 6a and 7 both have the potential to utilize H2, although through different forms of hydrogenase enzymes. Characterizing when / if these hydrogenases are expressed in Nitrosomonas cluster 6a and 7 AOB is the next step in determining their role within AOB.

Nitrogen Oxide Metabolism

- The metabolic process of ammonia oxidation produces the highly reactive, NO2 , as an - end product. NO2 can be reduced to NO, which causes nitrosative stress, through both biotic and abiotic processes (247). NO is also a potent greenhouse gas and the precursor to N2O, another - potent greenhouse gas (70, 73). To deal with NO2 as well as NO, the genomes of Nitrosomonas cluster 6a and 7 AOB encode different inventories of genes related to nitrogen oxide metabolism (Table 7) (2, 116, 119, 120). All 8 AOB genomes investigated in this study, and all characterized AOB genomes to date, encode the copper-containing nitrite reductase (nirK) (Table 7) (2, 116, 118-120). However, in all Nitrosomonas cluster 6a AOB genomes, nirK is encoded as a

46# # Table 7. Nitrogen oxide metabolism related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes.

Cluster 6a Cluster 7 N. N. Is791 Is341 JL21 AL2122 GH22 HPC101 oligotropha eutropha3 Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Locus Tag: Locus Tag: ABD07 Nit79A3 ABF85 ABF87 NAL212 Tag: Neut ABF86 ABO04 Nitrite Reductase nirK 141 2335 2040 1704 2392 - - - Singleton nirK gene Cluster - - - - - + + + Nitrite Reductase nirK - - - - - 1403 1476 69 ncgC - - - - - 1404 1475 70 ncgB - - - - - 1405 1474 71 ncgA - - - - - 1406 1473 72 nsrR - - - - - 1407 1472 73

Cytochrome c oxidase aa (NO 3 - - - - - + + + reductase sNOR) Subunit I (COG 843) - - - - - 1875 1808 1257 Subunit II (COG 1622) - - - - - 1874 1809 1256 senC - - - - - 1876 1807 1258

cNorCBQD - - - + + + + - norC - - - 2547 538 521 523 - norB - - - 2548 539 520 522 - norQ - - - 2549 540 519 521 - norD - - - 2550 541 518 520 -

Cytochrome c’ beta 1817 363 837 813 3151 1345 711 259 Cytochrome P460 598 1628 834 385 896 132 873 2148 Is79= Nitrosomonas sp. Is79, Is341= Nitrosomonas sp. Is341, JL21= Nitrosomonas sp. JL21, AL212= Nitrosomonas sp. AL212, GH22= Nitrosomonas sp. GH22 and HPC101= Nitrosomonas sp. HPC101. - = Not detected 1) Bollmann et al., 2013 2) Suwa et al., 2011 3) Stein et al., 2007

47# # singleton gene, where as in Nitrosomonas cluster 7 AOB nirK is found as part of a conserved gene cluster with other genes related to nitrosative stress (Table 7) (2, 116, 118-120).

- Aside from the reduction of NO2 to NO under hypoxic or anoxic conditions as part of the denitrification pathway, NirK has also been identified as an essential enzyme for efficient aerobic ammonia oxidation (70). During ammonia oxidation it is predicted to shuttle electrons

- onto NO2 to prevent the build up of toxic hydroxylamine, which is a necessary intermediate in aerobic ammonia-oxidation (70). The involvement of NirK in efficient ammonia oxidation and not just denitrification as suggested by Kozlowski et al., 2014, may explain why nirK is encoded as a singleton gene in the genomes of Nitrosomonas cluster 6a AOB and not within the conserved multi-gene cluster found in Nitrosomonas cluster 7 AOB genomes. This conserved

- multi-gene cluster also contains the NO2 or NO responsive transcription factor nsrR (236), which has been shown to transcriptionally regulate genes involved in nitrosative stress response

- (244). The absence of a master regulator for NO2 and nitrosative stress in the genomes of Nitrosomonas cluster 6a AOB, suggests that Nitrosomonas cluster 6a AOB have a different

- response to NO2 and NO than Nitrosomonas cluster 7 AOB (Table 7). A different cellular - response to NO2 and nitrosative stress may explain the observed sensitivity of Nitrosomonas - cluster 6a AOB but not Nitrosomonas cluster 7 AOB to high concentrations of NO2 (Fig. 4C). The genomes of all 5 Nitrosomonas cluster 6a AOB in this study also lacked the heme- copper NO reductase, sNOR, that is encoded in all 3 of the Nitrosomonas cluster 7 AOB genomes characterized (Table 7). In addition, the NO reductase, (norCBQD), genes were present in some but not all Nitrosomonas cluster 6a or 7 AOB genomes (Table 7). The absence of both forms of NO reductases in the genomes of N. oligotropha, Nitrosomonas sp. Is79, and Nitrosomonas sp. Is341 (Table 7) suggests that these 3 Nitrosomonas cluster 6a AOB can not reduce NO to N2O, or utilize a novel and to date uncharacterized enzyme. As eutrophication continues to spread into historically oligotrophic environments, AOB community succession from Nitrosomonas cluster 6a AOB to Nitrosomonas cluster 7 AOB, may lead to increased N2O emissions. However, confirmatory PCRs are needed to ensure the absence of both types of NO reductases in the draft genome sequences of both N. oligotropha and Nitrosomonas sp. Is341. All AOB presented here do encode cytochrome c’-beta and cytochrome P460 which are involved in

NO detoxification (Table 7) (245). The ability of Nitrosomonas cluster 6a AOB to produce N2O has not yet been investigated but is of interest due to the lack of NO reductases that are found in

48# # the genomes of Nitrosomonas cluster 6a AOB (Table 7). The overall lack of genes to deal with

- NO2 toxicity and nitrosative stress present the genomes of Nitrosomonas cluster 6a AOB (Table - 7), may explain their observed sensitivity to NO2 (Fig. 4C). On a similar note, the abundance of nitrogen oxide metabolism genes present in the genomes of Nitrosomonas cluster 7 AOB may

- explain why they are insensitive to high NO2 concentrations (Fig. 4C).

Carbon fixation Nitrosomonas cluster 6a and 7 AOB are most often found in freshwater aquatic environments (141) where CO2 concentrations are in constant fluctuation. In freshwater aquatic environments CO2 concentrations can be largely affected by pH as well as the growth of autotrophic microorganisms and stratification or mixing events (237). Nitrosomonas cluster 6a and 7 AOB each have separate physiological adaptations to adapt to varying CO2 concentrations (Table 8). The genomes of Nitrosomonas cluster 6a AOB encode 2 different Forms of RuBisCO enzymes, a Form IA green-like (high affinity) RuBisCO and a Form IC red-like (low affinity) RuBisCO (Table 8). While the genomes of Nitrosomonas cluster 7 AOB only encode a single Form IA green-like (high affinity) RuBisCO (Table 8). It is not uncommon for Alpha-, Beta- or GammaProteobacteria to encode more than one Form of RuBisCO (238). To date, no Form II RuBisCO enzymes have been identified in any characterized AOB genomes (2, 116-120). Form

II RuBisCO enzymes have a lower discrimination rate for O2 as an alternative substrate, compared to Form I RuBisCO enzymes (238). Because of this, Form II RuBisCO enzymes are often found in microorganisms living in low O2 and high CO2 environments (238). As aerobic microorganisms that require O2 as a co-substrate for ammonia oxidation, a low O2 and high CO2 environment is not favorable for AOB. Therefore, differentially expressing 2 Form I RuBisCO enzymes with different affinities for CO2 may be a more efficient scenario than expressing both a Form I and a Form II RuBisCO enzyme. Although the genomes of Nitrosomonas cluster 7 AOB do not encode a high affinity RuBisCO, the low affinity RuBisCO is often part of a large gene cluster that includes carboxysome biosynthetic genes (Table 8) (116, 119, 232, 233). The genome of Nitrosomonas sp. GH22 and all of the Nitrosomonas cluster 6a AOB genomes do not appear to encode any carboxysome related inventory (Table 8). Without the ability to differentially regulate two Forms of RuBisCO enzymes, like Nitrosomonas cluster 6a AOB, Nitrosomonas cluster 7 AOB appear

49# # Table 8. Carbon metabolism related genetic inventory of Nitrosomonas cluster 6a and 7 AOB genomes.

Nitrosomonas Cluster 6a Nitrosomonas Cluster 7 N. N. Is791 Is341 JL21 AL2122 GH22 HPC101 oligotropha eutropha3 Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: Locus Tag: ABD07 Nit79A3 ABF85 ABF87 NAL212 Neut ABF86 ABO04 RuBisCO From IA (green like) + + + + + + + + Carboxysome gene cluster - - - - - + - + Von Willebrand factor type A 1947 1252 2478 2495 869 816 1938 851 Nitric oxide reductase NorQ 1946 1253 2479 2495 870 815 1937 850 protein Ribulose bisphosphate 1945 1254 2480 2496 871 805 2406 849 carboxylase small chain Ribulose bisphosphate 1944 1255 2481 2497 872 804 2407 848 carboxylase large chain LysR family transcriptional 1943 1256 2482 2498 873 803 2408 847 regulator

RuBisCO From IC (red like) + + + + + - - - LysR family transcriptional 2895 3234 783 2924 2941 - - - regulator Ribulose bisphosphate 2896 3235 782 2923 2942 - - - carboxylase large chain Ribulose bisphosphate 2897 3236 781 2922 2943 - - - carboxylase small chain CbbX protein 2898 3237 780 2921 2944 - - - Hypothetical protein 2899 3238 779 2920 2945 - - - Ketose-bisphosphate aldolase 2900 3239 - - 2946 - - - Is79= Nitrosomonas sp. Is79, Is341= Nitrosomonas sp. Is341, JL21= Nitrosomonas sp. JL21, AL212= Nitrosomonas sp. AL212, GH22= Nitrosomonas sp. GH22 and HPC101= Nitrosomonas sp. HPC101. - = Not detected 1) Bollmann et al., 2013 2) Suwa et al., 2011 3) Stein et al., 2007 !

! 50! to utilize carboxysomes to enhance carbon fixation rates in low CO2 conditions. The ultilization of CO2 concentrating mechanisms such as carboxysomes is common among bacteria inhabiting environments that experience low CO2 concentrations (238, 239). Overall, Nitrosomonas cluster 6a and 7 AOB utilize different adaptive traits to handle the fluctuating CO2 concentrations they encounter (Table 8). !

! 51! Conclusions This study physiologically and genomically compared multiple AOB species and isolates belonging to both Nitrosomonas cluster 6a and 7 (Table 1; Fig. 3). These strains were

+ specifically targeted as representatives of AOB, which are adapted to low and high NH4 concentrations respectively. The intent was to identify and link conserved genomic inventory to the observed different physiological adaptations of these AOB. The presence or absence of genomic inventory related to physiological traits such urea utilization (Table 6) and the

- sensitivity of Nitrosomonas cluster 6a AOB but not Nitrosomonas cluster 7 AOB to high NO2 concentrations (Table 7) correlated well with observed physiological characteristics (Fig. 4C, F). In addition, this study identified several conserved traits that differed between the genomes of Nitrosomonas cluster 6a and 7 AOB. These traits are related to the flexibility of Nitrosomonas cluster 6a and 7 AOB to capture O2 (Table 5) and CO2 (Table 8) over a range of concentrations. Together, these traits shed light on the conserved adaptations that differ between Nitrosomonas cluster 6a and 7 AOB. Traits such as urea utilization, H2 utilization and carboxysome production appeared to be species-specific and were not conserved among all Nitrosomonas cluster 6a or 7 AOB (Table 6 and 8). This finding highlights the fact that although closely related species often share many of the same physiologic characteristics, even closely related species can have unique characteristics. Identifying conserved genomic inventory that all AOB have in common is essential to determine the enzymatic machinery essential for aerobic NH3 oxidation. In addition, determining physiological characteristics that are conserved among groups of AOB as well as species-specific adaptations is crucial to begin investigating how AOB community succession may affect overall ecosystem function. A strong example of the power of this approach is the disparity in nitrogen oxide metabolism inventory between Nitrosomonas cluster 6a and Nitrosomonas cluster 7 AOB. As historically oligotrophic ecosystems are subject to eutrophication, AOB community

+ succession based on tolerance to different NH4 concentrations may affect the production of nitrogenous GHGs such as N2O. Often, microorganisms are broadly labeled by their function such as ammonia-oxidizer; but as more microbial genomes are sequenced, we are discovering that closely related species within the same functional groups can have an array of different metabolic capabilities.

! 52! CHAPTER 2

The effect of bacterial community members on the proteome of the ammonia-oxidizing bacterium Nitrosomonas sp. Is79

Christopher J. Sedlacek, Susanne Nielsen, Kenneth D. Greis, Wendy D. Haffey, Niels Peter Revsbech, Tomislav Ticak, Hendrikus J. Laanbroek, and Annette Bollmann

Submitted to the journal of Applied and Environmental Microbiology

! 53! Abstract

Microorganisms in the environment live in diverse microbial communities and not as the often-studied pure cultures. Characterizing microbial interactions is essential to understand their performance in both natural and engineered environments. In this study we investigate how the presence of nitrite-oxidizing bacteria (NOB) and heterotrophic bacteria affect growth and the proteome of the chemolithoautotrophic ammonia-oxidizing bacteria (AOB) Nitrosomonas sp. Is79. We investigated Nitrosomonas sp. Is79 as axenic culture, defined co-cultures of Nitrosomonas sp. Is79 with Nitrobacter winogradskyi and selected heterotrophic bacteria, and the enrichment culture G5-7 consisting of Nitrosomonas sp. Is79, heterotrophic bacteria and N. winogradskyi. Batch culture growth experiments showed that N. winogradskyi and the heterotrophs had positive effects on the growth of Nitrosomonas sp. Is79. An isobaric tag for relative quantification (iTRAQ) LC MS/MS proteomics approach was used to investigate the effect of N. winogradskyi and the co-cultivated heterotrophic bacteria on the proteome of Nitrosomonas sp. Is79. The proteome of Nitrosomonas sp. Is79 was altered differently in the presence of N. winogradskyi and heterotrophic bacteria. In co-culture with N. winogradskyi, the abundance of the proteins related to the oxidative stress response increased in the periplasm but decreased in the cytoplasm. The abundance of ATP synthase proteins also declined in the AOB- NOB co-culture. When co-cultured with heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation pathway increased, while the abundance of proteins related to amino acid synthesis and metabolism decreased. We conclude that in the presence of N. winogradskyi and heterotrophic bacteria Nitrosomonas sp. Is79 exhibited a decrease in the production of proteins associated with intracellular oxidative stress response in combination with more efficient energy generation from ammonia oxidation through NADPH production.

! 54! Introduction

- - Nitrification, the oxidation of ammonia (NH3) to nitrate (NO3 ) via nitrite (NO2 ), is a microbially driven two-step process within the global nitrogen cycle (26, 131, 201). The first

- step, the oxidation of NH3 to NO2 is carried out aerobically by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) (95, 103, 105, 141). AOB are chemolithoautotrophic bacteria that generate their energy through ammonia oxidation and fix carbon through the Calvin cycle (105, 131). The second step of nitrification is performed by

- - nitrite-oxidizing bacteria (NOB), which generate energy from the oxidation of NO2 to NO3 (27, 28, 154). Betaproteobacterial ammonia oxidizers are comprised of the genera Nitrosomonas and Nitrosospira. Within the genus Nitrosomonas, AOB form several clusters that can be distinguished by ecophysiological characteristics such as affinity for NH3, tolerance to salt or - NO2 (80,141,201). Nitrosomonas cluster 6a is comprised of freshwater AOB adapted to low NH3 concentrations that can be found in oligotrophic environments (164, 217, 248, 249). Nitrosomonas sp. Is79, a member of Nitrosomonas cluster 6a, was isolated from freshwater

+ sediment and is adapted to low ammonium (NH4 ) concentrations (2, 143, 164). In the environment, microorganisms do not exist in isolation, but live in complex interacting communities. Within microbial communities negative interactions such as competition or predation can be detected along with mutually beneficial or synergistic interactions such as cross feeding, co-metabolism or cell-to-cell communication (5, 6, 11, 12). Photo- as well as chemolithoautotrophic microbes interact with heterotrophic microbes in many different environments. For example, macro- and microalgae live with specific heterotrophic microbial communities, which produce and exchange nutrients, enzymes and bioactive compounds that result in increased community productivity (250, 251). AOB interact positively with NOB and heterotrophic bacteria (7-10, 147). The positive effect of NOB on the growth of

- AOB is in large part due to the removal of NO2 from the environment by NOB, as it inhibits AOB growth (147, 154, 252). In addition, a study investigating the transcriptome of Nitrosomonas europaea identified more efficient carbon assimilation and changes in electron transport as possible positive effects of co-cultivation with the NOB Nitrobacter winogradskyi (147). AOB provide organic carbon as substrate in the form of soluble microbial products (SMP) to co-cultured heterotrophic bacteria (7, 8). The positive growth response of AOB to the presence

! 55! of heterotrophic bacteria could be due to a variety of compounds that may be secreted by the heterotrophic bacteria such as organic compounds (253-256), acyl-homoserine lactones (243, 258) or siderophores (9). Studies on the interactions between AOB and heterotrophic bacteria have mainly focused on the members, structure and functionality of the heterotrophic bacterial communities (7, 8). To date it has not been elucidated how the proteome of AOB respond to the presence of co-cultured heterotrophic bacteria. To our knowledge, only a few studies have been conducted focusing on the transcriptome or proteome of AOB. These studies have mainly utilized N. europaea or Nitrosomonas eutropha, two fast growing AOB often found in and adapted to nutrient rich environments (72, 147, 259- 262). Studies focusing on the analysis of proteomes have concentrated on differing environmental or physiologic growth conditions such as starvation (262), heavy-metal (261), biofilm formation (72) and the effect of nitrogen dioxide (NO2) or anoxia (260). Here we present a study investigating the effect of N. winogradskyi as well as the co- cultivated heterotrophic community in the enrichment culture G5-7 on the growth physiology of the ammonia oxidizer Nitrosomonas sp. Is79. We employed a combination of growth experiments and isobaric tag for relative quantification (iTRAQ) LC MS/MS proteomics to determine whether the proteome of Nitrosomonas sp. Is79 is influenced by the presence of N. winogradskyi and heterotrophic bacteria.

! 56! Material and Methods Cultures and media Cultures: We used the following cultures derived from the original AOB enrichment culture G5- 7 (143, 164): (1) AOB isolate Nitrosomonas sp. Is79 (2), (2) the AOB enrichment culture G5-7 itself, which contains the AOB Nitrosomonas sp. Is79, N. winogradskyi, and co-cultivated heterotrophic bacteria, and (3) newly isolated heterotrophic bacteria from the enrichment culture G5-7. We also used the nitrite oxidizer N. winogradskyi (ATCC 25391).

Medium to cultivate AOB and NOB: The AOB mineral salts (MS) medium used for AOB growth experiments contained 10mM NaCl, 1mM KCl, 1mM CaCl2!2H2O, 0.2mM MgSO4!7H2O and 1mL liter-1 trace elements solution (169, 211). Batch cultures were buffered with HEPES, which

+ was added in a 4-fold molar ratio to the NH4 concentration and the pH was adjusted to 7.8 before autoclaving (169). After autoclaving, sterile KH2PO4 was added to a final concentration of

0.4mM. N. winogradskyi was cultivated in NOB-MS medium that contained 14.5mM NaNO2, -1 8.6mM NaCl, 1.1mM KH2PO4, 0.2mM MgSO4!7H2O, 0.03mM CaCO3 and 1mL liter trace elements solution with pH 7.8 (211, 249).

Media to cultivate the heterotrophic bacteria: LB medium (Sigma Aldrich, St Louis, MO, USA), R2A medium (264) and spent AOB medium were used to isolate and cultivate heterotrophic bacteria. Spent AOB medium consisted of 25% sterile filtered medium from an outgrown Nitrosomonas sp. Is79 culture, 75% AOB-MS medium and either 0.01g/l casamino acids or 0.01g/l yeast extract. If necessary, media was solidified with 1.5% Bacto agar (Difco, Becton, Dickinson and Company, Sparks, MD, USA).

Isolation of heterotrophic bacteria from the enrichment culture G5-7: Late log phase G5-7

+ culture was serially diluted with MS medium without NH4 and inoculated onto agar plates containing either LB, 0.1xLB, R2A and spent AOB medium. Individual colonies were selected based on colony morphology, sub-cultured and further maintained on R2A agar plates. Colony PCR and 16S rDNA sequencing were used to identify the isolated colonies.

! 57! Contamination tests: Axenic cultures of Nitrosomonas sp. Is79 and N. winogradskyi were inoculated into LB medium (0.1x) and incubated at 27oC for two weeks to test the purity of the autotrophic cultures. Cultures exhibiting growth of heterotrophic bacteria were discarded.

Kinetic studies

Apparent Michaelis constant (Km(app)): Nitrosomonas sp. Is79 as a pure culture, in co-culture with N. winogradskyi and the enrichment culture G5-7 were grown as batch cultures with continuous stirring and bubbling at 25oC in the dark. Late logarithmic growth phase cultures (1 L) were

o + harvested by centrifugation (20 min, 30,000 x g, 4 C), washed and re-suspended in NH4 -free + MS medium (10 mL). NH4 free MS medium (3.6 mL) was mixed with the concentrated cell + + suspension (0.4 mL). Concentrated NH4 solution was added to obtain final NH4 concentrations - - - - between 20 and 1000 µM. Initial NO2 /NO3 production was followed by a NO2 /NO3 biosensor - - and used to calculate NO2 /NO3 production rates (175). Michaelis-Menten kinetics was applied to calculate the apparent half saturation constant of ammonia oxidation (Km(app)) (265).

Growth experiments

Half saturation constant of growth (Ks): The growth rate of Nitrosomonas sp. Is79 as a pure culture, in co-culture with N. winogradskyi and in the enrichment culture G5-7 was determined

+ across a range of initial NH4 concentrations (0.25 – 5 mM). The cultures were inoculated with 10% (v/v) of a late logarithmic phase culture, incubated at 25°C in the dark and shaken at 120

+ - - rpm. Samples were taken regularly to determine NH4 , NO2 and NO3 concentrations. Growth - - - rates were determined by calculating the slope of the log transformed NO2 or NO2 / NO3 - - concentrations against time, assuming a correlation between NO2 /NO3 production and growth

(169, 212). Monod kinetics was applied to determine the half saturation constant of growth (Ks) of Nitrosomonas sp. Is79 in the different cultures (265).

Growth experiments of Nitrosomonas sp. Is79 with heterotrophic bacteria and N. winogradskyi:

+ All growth experiments were conducted in AOB-MS medium with 1mM NH4 . Cultures were inoculated with 1% (v/v) late logarithmic phase Nitrosomonas sp. Is79 and one or more of the following: 0.2% (v/v) R2A media, 0.2% (v/v) culture of heterotrophic bacteria cultivated in R2A or 1% (v/v) N. winogradskyi culture. In addition we used the enrichment culture G5-7 and G5-7

! 58! minus the NOB. The NOB in the enrichment culture G5-7 were inhibited and subsequently eliminated from the culture by the addition of 10mM NaClO3 (252). Heterotrophic bacterial cultures were pre-cultured in liquid R2A for 2 days prior to inoculation. All 10 heterotrophic bacteria were combined into a mixed culture, which was added to Nitrosomonas sp. Is79 to resemble as well as possible composition of the original G5-7 culture. When the cultures had

+ consumed the NH4 they were transferred to fresh AOB-MS medium (1% v/v). This transfer was repeated once more resulting in three growth cycles. Samples were taken regularly during growth

o - - cycle 1 and 3 and stored at -20 C to determine NO2 /NO3 production and the growth rate of Nitrosomonas sp. Is79. Following the last growth cycle, co-cultures were plated on R2A agar. Colony PCR and sequencing were used to confirm the identity of the heterotrophic bacteria.

Chemostat setup: Chemostats with a working volume of 5 L were assembled and autoclaved

+ with 4.5 L of unbuffered AOB-MS medium with 5 mM NH4 . Once cooled the temperature was adjusted to 27oC with a temperature blanket, stirring (300 rpm) and bubbling (500 mL min-1) with 0.2 μm sterile filtered atmospheric air were started and the oxygen sensor was calibrated.

Sterile KH2PO4 was added to a final concentration of 0.4 mM and the pH was adjusted to 7.8 with sterile 3% NaCO3. The chemostats were incubated in the dark and inoculated with late logarithmic phase batch cultures (0.5 L) of Nitrosomonas sp. Is79, Nitrosomonas sp. Is79 co- cultured with N. winogradskyi or the enrichment culture G5-7. Samples (20 mL) were taken in regular intervals, filtered through a polycarbonate filter (0.2 µm, Whatman Nuclepore) and stored at -20oC for chemical analysis. Contamination tests were conducted each time a sample

+ was taken. When the initial NH4 in the chemostat was consumed, unbuffered AOB-MS medium + -1 containing 5 mM NH4 was added at a dilution rate of approximately 1.25 L day (growth rate = 0.25 day-1). Chemostats were run for 12 days, which corresponds to 3 volume changes. Total cell biomass was harvested by centrifugation (20 min, 22,000 x g, 4oC). The cell pellets were washed

o in phosphate buffer (20 mM KH2PO4, pH=8), centrifuged (20 min, 28,000 x g, 4 C) and stored at -80oC for proteomic analysis.

+ - - Chemical analysis: Colorimetric assays were used to determine NH4 , NO2 and NO3 concentrations in cell free supernatants (169, 213, 214, 267).

! 59! Molecular analysis Colony PCR and sequencing: Colony cell material was used as template for PCR. The 16S rRNA gene was amplified with the eubacterial primers 27F and 1492R (Table 9) using the GoTaq Green Master Mix (Promega, Madison, WI) (268). The PCR products were purified using the Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI) and sequenced with the primers 357F and 518R (269) using a BigDye Terminator cycle sequencing kit on an Applied Biosystems 3730 DNA analyzer (Life Technology Corporation, Carlsbad, CA, USA) at the Center for Bioinformatics and Functional Genomics (CBFG) at Miami University. The sequences were edited in 4Peaks (A. Griekspoor and T. Groothuis, The Netherlands Cancer Institute). ARB was used to align the sequences and to determine the closest cultures relative (216).

Molecular analysis of the heterotrophic community in the enrichment culture G5-7: The enrichment culture G5-7 (50 ml) was filtered onto a 0.2 µm nucleopore filter (Whatman Nuclepore). DNA was extracted with the FastDNA Spin Kit for Soil (MP Biomedicals, Solon OH) according to the manufacturers recommendations with the following modifications. Filters were homogenized using a bead beater (Biospec Products, Bartlett, OK) three times for 30s at 4800rpm. Samples were stored on ice for 10 min between bead beating steps and centrifuged (15 min, 28,000 x g, 4oC) after the third round of bead beating.

Next generation sequencing: DNA from the enrichment culture G5-7 was amplified in triplicate with Illumina compatible indexed primers designed to amplify the V4 region of the 16S rRNA gene (515F-806R) (270). Since the sample was part of a larger sequencing project a 12 base barcode was incorporated in the reverse primers (270, 271). The PCR products were quantified using a Sybr Green I dsDNA assay and the concentration was measured on a NanoDropTM 3300 Fluorospectrometer (Thermo Fisher Scientific, Wilmington, DE, USA). All PCR products were mixed in equal ratio and sequenced in a MiSeq (Illumina, San Diego, CA, USA) at the CBFG at Miami University.

Analysis of the Illumina sequence data: The sequences were processed by the software package MiSeq Reporter into two files: sequences and barcodes. The sequences were split based on the

! 60! Table 9. Primers used for amplification and sequencing

Primer Amplification (Weisburg et al., 1991) 27F: 5’-AGA GTT TGA TCC TGG CTC AG-3’ 1492R: 5’-GGT TAC CTT GTT ACG ACT T-3’ Sequencing (Muyzer et al., 1993) 357F: 5’-CCT ACG GGA GGC AGC AG-3’ 518R: 5’-ATT ACC GCG GCT GCT GG-3’

! 61! barcodes, quality filtered with a minimum quality score of 25, truncated to 150bp and exported as sequence files (fasta) using the software package QIIME (258). The operational taxonomic units (OTUs) were picked against the Greengenes database “gg_13_8_otus” with 97% similarity. Since there might be an increased diversity in pyrosequencing libraries due to sequencing errors and to focus on the dominant sequences all OTUs with an abundance below 10 were eliminated. The taxonomic affiliation of the sequences was determined and representative sequences of each OTU were aligned to the ARB database to determine the similarity to the isolated heterotrophic bacteria. The analysis of the community focused on the heterotrophic bacteria in the community.

Protein preparation and analysis Total cell protein quantification: Wet cell pellets (0.033-0.08 g) were suspended in phosphate buffer (500 µL, 20 mM KH2PO4, pH 8.0), combined with 1 g of 0.1 mm zirconia/silica beads and bead beat three times for 30 s at 4800 rpm. Samples were stored on ice for 10 min between each round and centrifuged (20 min, 28,000 x g, 4oC) after the third time. The supernatant was removed and stored at -80oC. The protein concentration of the whole cell extracts was quantified colorimetrically by Bradford reagent and bicinchoninic acid assay reagent according to the manufacture’s guidelines. Normalized whole cell protein extracts (15 μg) were prepared with 1X Laemmli buffer (273), heated (5 min, 95oC) and briefly centrifuged prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples and standards were run on a discontinuous 15% (Bis-Tris) SDS-PAGE gel (http://openwetware.org/wiki/Sauer:bis- Tris_SDS-PAGE%2C_the_very_best) in High-MW running buffer (50 mM MOPS, 50 mM Tris, 5 mM EDTA, 0.5% SDS with 5 mM sodium bisulfite at pH 8.0) at 100 V. The SDS-PAGE gel was stained while rocking overnight at 23oC in Coomassie blue stain and afterwards destained (273). The gel was imaged with a VersaDoc Imaging System and converted to gray scale with PDQuest (Bio-Rad Laboratories).

Solubilization and isolation of peptides: Biological replicates of cell lysates from Nitrosomonas sp. Is79, Nitrosomonas sp. Is79 co-cultured with N. winogradskyi, and enrichment culture G5-7 were solubilized in Laemmli gel buffer with heating to 110oC for 10 min. 50 ug of each sample (6 samples total) were loaded onto separated lanes of two 1D, 4-12% MOPS 1.0 mm mini gel, then electrophoresed for 15 min which was just long enough for the proteins to enter the gel. The

! 62! gel region containing the proteins (about 1.5 cm x 2.5 cm) was cut from the gel and subjected to in gel trypsin digestion and subsequent recovery of peptides as described previously (274). iTRAQ labeling: The Nitrosomonas sp. Is79 control samples were divided in half such that technical replicates were available for both controls. Two comparative groups were tagged using the 4-plex iTRAQ reagents following the vendor (AB Sciex) instructions and as described previously (274). In set one the 116 and 117 reporter tags were used for the isolated peptides of Nitrosomonas sp. Is79 co-cultured with N. winogradskyi samples (3 and 4) while the 114 and 115 reporter tags were used for the Nitrosomonas sp. Is79 control samples (1 and 2). In set two the 116 and 117 reporter tags were used for the isolated peptides of the enrichment culture G5-7 samples (5 and 6) while the 114 and 115 reporter tags were used for the Nitrosomonas sp. Is79 control samples (1 and 2). After labeling, the samples were mixed together in equal quantities for subsequent separation, identification and quantitative analysis.

Nano-electrospray liquid chromatography couple tandem mass spectrometry (nLC-ESI-MS/MS): nLC-ESI-MS/MS analyses were performed on a TripleTOF 5600 plus (ABSciex, Toronto, On, Canada) attached to an Eksigent (Dublin, CA) nanoLC.ultra nanoflow system. 2.0 ug of total protein from each 4-plex mixture was loaded (via an Eksigent nanoLC.as-2 autosampler) onto an IntegraFrit Trap Column (outer diameter of 360 µm, inner diameter of 100, and 25 µm packed bed) from New Objective, Inc. (Woburn, MA) at 2 µl/min in formic acid/H2O 0.1/99.9 (v/v) for 15 min to desalt and concentrate the samples. For the chromatographic separation of peptides, the trap-column was switched to align with the analytical column, Acclaim PepMap100 (inner diameter of 75 µm, length of 15 cm, C18 particle sizes of 3 µm and pore sizes of 100 Å) from Dionex-Thermo Fisher Scientific (Sunnyvale, CA). The peptides were eluted using a varying mobile phase (MP) gradient from 95% phase A (FA/H2O 0.1/99.9, v/v) to 40% phase B (FA/ACN 0.1/99.9, v/v) for 70 mins, from 40% phase B to 85% phase B for 5 mins and then keeping the same MP-composition for 5 more minutes at 300 nL/min. The nLC effluent was ionized and sprayed into the mass spectrometer using NANOSpray® III Source (ABSciex, Toronto, On, Canada). Ion source gas 1 (GS1), ion source gas 2 (GS2) and curtain gas (CUR) were respectively kept at 15, 0 and 30 vendor specified arbitrary units. Interface heater temperature and ion spray voltage was kept at 150oC, and at 2.3 kV respectively. Mass

! 63! spectrometer method was operated in positive ion mode set to go through 2916 cycles for 80 minutes, where each cycle performing one TOF-MS scan type (0.25 secs accumulation time, in a 350 to 1250 m/z window) followed by thirty information dependent acquisition (IDA)-mode MS/MS-scans on the most intense candidate ions having a minimum 150 counts. Each product ion scan was operated under vender specified high-sensitivity mode with an accumulation time of 0.06 secs and a mass tolerance of 50 mDa. Former MS/MS-analyzed candidate ions were excluded for 13 secs after its first occurrence, and data were recorded using Analyst®-TF (v.1.6) software.

Data Analyses: Searches from the nLC-MS/MS were accomplished using ProteinPilot software (version 4.5, revision 1656) that utilizes Paragon algorithm (ver 4.5.0.0, 1654) against database created of N. winogradskyi and Nitrosomonas sp. Is79 protein sequences supplemented with common contaminating proteins (human keratins, porcine trypsin, etc) for a total of 12,988 proteins searched. ProteinPilot search parameters included all biological modification as variable modifications with carboxyamidomethyl cysteine was used as a fixed modification. Gel-based ID was selected as a special factor. The precursor mass tolerance is automatically set by the ProteinPilot software based on the instrument type used—in this case the instrument type selected is a quadrupole-Tof instrument. Data normalization across all iTRAQ tag channels was accomplished in the ProteinPilot software using the bias correction function. This function evaluated the relative ratio of the iTRAQ tags across all the peptides identified and normalizes the ratios such that the collective ratio of all the tags is 1:1:1:1, prior to calculating relative differences among individual peptides/protein. The output files for the ProteinPilot database search (*.group file) contained a summary statistics page, the peptide identification tables, protein identification tables and relative quantitation data from the iTRAQ reporter ions from each peptide all of which was exported as an Excel spreadsheets for further statistical analysis using the ProteinPilot Descriptive Statistics Template (PDST, ver 3.005pB) (http://www.absciex.com/Documents/Downloads/Literature/ProteinPilot-Descriptive-Stats- Template-MassSpec-1910211-01.pdf). The PDST is a mathematical Excel template that processes the relative quantitation data among the sample sets and provides statistical probabilities related to the confidence of the protein identification in relationship to an inverse (decoy) protein database, and provide p values regarding the significance of relative quantitation

! 64! of the 4 reporter ions for each protein. For protein identification and quantitative profiling reported in the data tables, a minimum fold-change of 1.5x from a least 2 peptides with a p-value of greater than 0.01 was required.

! 65! Results Community analysis of the heterotrophic bacteria in enrichment culture G5-7. Next generation sequencing of the heterotrophic community of the enrichment culture G5- 7 revealed a total of 19 unique OTU’s (Table 10). Based on reads, the community was comprised of Alphaproteobacteria (70.3%), Gammaproteobacteria (24.42%), Betaproteobacteria (2.46%) and Bacteriodetes (2.82%). Ten (10) unique heterotrophic bacteria were isolated from the enrichment culture G5-7 (Table 11). All 10 isolates were detected in the next generation sequencing library of the enrichment culture G5-7 and their abundance made up 87.7% of the sequences of the co-cultured heterotrophic bacteria. All 10 of the isolates are members of the Proteobacteria (Table 11).

Ammonia-oxidizing activity and growth characteristics of Nitrosomonas sp. Is79.

- - The NO2 /NO3 production of Nitrosomonas sp. Is79 exhibited a longer lag phase and increased slower in pure culture compared to either the co-culture with N. winogradskyi or as part of the enrichment culture G5-7 (Fig. 5). The growth rate of Nitrosomonas sp. Is79 (0.023 ± 0.003 h-1) increased when grown in co-culture with N. winogradskyi (0.037 ± 0.002 h-1) and was highest when grown as part of the enrichment culture G5-7 (0.055 ± 0.002 h-1) (Fig. 5). The half saturation constant of growth (Ks) decreased when Nitrosomonas sp. Is79 was grown in co- culture with N. winogradskyi or as part of the enrichment culture G5-7 (Table 12). However, the

+ apparent Michaelis constant (Km(app)) of NH4 consumption remained constant in all three cultures (Table 12).

The effect of N. winogradskyi and the newly isolated heterotrophic bacteria on the growth rate of Nitrosomonas sp. Is79. The growth rate of Nitrosomonas sp. Is79 was determined in a variety of artificially designed co-cultures containing different combinations of the newly isolated heterotrophic bacteria as well as the presence or absence of N. winogradskyi over the course of 3 growth cycles (2 culture transfers). In general the growth rate of Nitrosomonas sp. Is79 increased in the presence of N. winogradskyi and the heterotrophic isolates (Fig. 6). No negative effects were detected. The growth rate of Nitrosomonas sp. Is79 as part of the enrichment culture G5-7 was comparable to the artificial community of Nitrosomonas sp. Is79 with all 10 heterotrophic

! 66! Table 10. Phylogenetic affiliation of the heterotrophic bacteria present in the ammonia- oxidizing enrichment culture G5-7 determined by amplicon sequencing.

OTU # Phylogenetic affiliation Abundance [%] Corresponding isolate Alphaproteobacteria 1 Rhizobiales 4.45 G5-7 Is2; G5-7 Is28 ! 2 Bradyrhizobiaceae 29.90 G5-7 Is42 ! ! 3 Bradyrhizobium sp. 15.08 G5-7 Is42 4 Bosea sp. 4.10 5 Agrobacterium sp. 1.07 G5-7 Is17 6 Methylorhabdus sp. 8.74 G5-7 Is3 7 Hyphomicrobiaceae 3.82 8 Hyphomicrobium sp. 0.25 9 Rhodoplanes sp. 0.17 10 Phyllobacteriaceae 0.91 11 Phenylobacterium 1.31 G5-7 Is32 12 Sphingomonadales 0.51 G5-7 Is23 Betaproteobacteria 13 Beta-Proteobacteria 0.17 14 Pandoraea sp. 2.29 G5-7 Is22 Gammaproteobacteria 15 Pseudomonas sp. 23.93 G5-7 Is19 16 Moraxellaceae 0.05 17 Luteimonas 0.44 G5-7 Is39 Bacteroidetes 18 Sediminibacterium sp. 2.72 19 Flavobacterium sp. 0.10

! 67! Table 11. Phylogenetic affiliation of the bacterial isolates from the enrichment cultures G5- 7 and their abundance in the enrichment culture based on the sequence abundance in the 16S rRNA library.

G5-7 Closest cultured relative Similarity Sequence length Abundance in [%] [bp] G5-7 [%] 1 Alphaproteobacteria Is42 Afipia broomeae (U87759) 99.7 1099 44.8 Is3 Ancylobacter aquaticus (M62790) 99.0 625 8.7 Is2 Afipia genospecies 7 (U87773) 99.4 1089 4.4 (=Is28) 2 Is28 Mesorhizobium huakii (D12797) 99.8 1372 4.4 (=Is2) 2 Is32 Caulobacter fusiformis (AJ007803) 98.5 1082 1.3 Is17 Rhizobium radiobacter (M11223) 96.6 1071 1.1 Is23 Sphingomonas adhaesiva (D13722) 98.8 1100 0.5 Betaproteobacteria Is22 Pandoraea pnomenusa (AF139174) 99.8 1076 2.3 Gammaproteobacteria Is19 Pseudomonas putida (AF094743) 99.6 1137 23.9 Is39 Lysobacter antibioticus (AB019582) 95.7 1091 2.3 1 Abundance in the enrichment culture G5-7 based on the total reads assigned to OTU’s representing heterotrophic bacteria. 2 Based on the short read length in NGS Is2 and Is28 cluster together with OTU1 (Table 10).

! 68!

- - - Figure 5. NO2 or NO2 /NO3 production by Nitrosomonas sp. Is79 (Is79), Nitrosomonas sp. Is79 together with N. winogradskyi (Is79+NOB) and the enrichment culture G5-7 (G5-7) (mean ± SD, n=3).

! 69!

1000 Is79 Is79+NOB G5-7

M] 800

600 concentration [

- 400 3 /NO - 2 200 NO

0 0 50 100 150 200 time [h]

Figure 1: Nitrite or nitrite/nitrate production of Nitrosomonas sp. Is79 (Is79), Nitrosomonas sp. Is79 together with N. winogradskyi (Is79+NOB) and the enrichment culture G5-7 (G5-7) (mean ± SD, n=3).

! 70! Table 12. Half saturation constant of ammonia-oxidizing activity (Km(app)) and growth (Ks) of Nitrosomonas sp. Is79 in pure culture (Is79), in the presence of N. winogradskyi (Is79+NOB) and as part of the enrichment culture G5-7 (G5-7).

Is79 Is79+NOB G5-7 1 Km(app) [µM NH3] 3.45 ± 0.3 3.35 ± 0.5 3.45 ± 0.1 2 Ks [µM NH3] 17.9 ± 3.1 9.4 ± 1.9 8.2 ± 2.9 1 (mean ± range, n=2) 2 (mean ± SD, n=3)

! 71!

Figure 6. Growth of Nitrosomonas sp. Is79 in co-culture with heterotrophic bacteria isolated from the enrichment culture G5-7 and N. winogradskyi during different growth cycles (mean ± SD, n=3). A: during the first growth cycle and B: during the third growth cycle. Data presented as % of the growth rate of Nitrosomonas sp. Is79 alone, which is set to 100%. Nitrosomonas sp. Is79 was combined with all the heterotrophic isolates and N. winogradskyi in co-cultures and in a mixed culture with all heterotrophs with and without N. winogradskyi (All isolates and N. winogradskyi + all isolates). Finally Nitrosomonas sp. Is79 was tested as part of the enrichment culture G5-7 in the presence and absence of N. winogradskyi. R2A medium was added in place of a heterotrophic bacterium as a control.

! 72!

300 300 A B

250 250

200 200

150 150

100 100 Growth rate [% control] Growth rate [% control] 50 50

0 0 G5-7 G5-7 G5-7 Is3 G5-7 Is2 G5-7 Is3 G5-7 Is2 G5-7 Is42 G5-7 Is28 G5-7 Is32 G5-7 Is17 G5-7 Is23 G5-7 Is22 G5-7 Is19 G5-7 Is39 G5-7 Is42 G5-7 Is28 G5-7 Is32 G5-7 Is17 G5-7 Is23 G5-7 Is22 G5-7 Is19 G5-7 Is39 All isolates All isolates

Proteobacteria N. winogradskyi Proteobacteria N. winogradskyi R2A media control R2A media control G5-7 -N.winogradskyi G5-7 -N.winogradskyi

N. winogradskyi + all isolates N. winogradskyi + all isolates

Figure 2: Growth of Nitrosomonas sp. Is79 in co-culture with heterotrophic bacteria isolated from the enrichment culture G5-7 and N. winogradskyi during different growth cycles (mean ± SD, n=3). A: during the first growth cycle and B: during the third growth cycle. Data presented as % of the growth rate of Nitrosomonas sp. Is79 alone, which is set to 100%. Nitrosomonas sp. Is79 was combined with all the heterotrophic isolates and N. winogradskyi in co-cultures and in a mixed culture with all heterotrophs with and without N. winogradskyi (All isolates and N. winogradskyi + all isolates). Finally Nitrosomonas sp. Is79 was tested as part of the enrichment culture G5-7 in the presence and absence of N. winogradskyi. R2A medium was added in place of a heterotrophic bacterium as a control.

! 73! isolates and N. winogradskyi (Fig. 6).

Chemostat Growth.

+ Nitrosomonas sp. Is79 was grown with NH4 as the sole energy source and growth limiting substrate as a pure culture, in co-culture with N. winogradskyi or as part of the enrichment culture G5-7 in continuous cultures (Fig. 7). Once the cultures reached steady state, the

+ concentration of the growth limiting substrate, NH4 , in the chemostats was higher in the pure culture of Nitrosomonas sp. Is79 (115.94 µM) than the co-culture of Nitrosomonas sp. Is79 with N. winogradskyi (2.54 – 6.35 µM) or the enrichment culture G5-7 (0.92 – 1.88 µM) (Table 13).

Proteomics. SDS-PAGE of whole cell extracts from the 3 different chemostat cultures showed different protein banding patterns (Fig. 8). However, direct inferences about the Nitrosomonas sp. Is79 proteome cannot be made because each sample contained a different bacterial community. Isobaric tagging using iTRAQ reagents followed by LC MS/MS was used to determine the proteomic profile of Nitrosomonas sp. Is79 in all 3 chemostat cultures. In each case, protein abundance ratios were based on the relative abundance of iTRAQ tags as described in detail in the supplemental methods. The proteomic analysis focused on the proteins of Nitrosomonas sp. Is79 which changed in abundance more than 1.5 times relative to the pure culture (p>0.01) when grown in co-culture with N. winogradskyi or as part of the enrichment culture G5-7. A full list of detected proteins with changes in abundances can be found in the supplemental material (Table 14 and 15). Due to technical problems only 1 of the 2 Nitrosomonas sp. Is79 chemostat cultures (tagged with the 115 iTRAQ reagent) was analyzed and used for comparison to the biological replicates (2 of each) of Nitrosomonas sp. Is79 co-cultured with N. winogradskyi and G5-7 chemostat cultures.

The influence of N. winogradskyi on the proteome of Nitrosomonas sp. Is79. The abundance of 55 Nitrosomonas sp. Is79 proteins exhibited an increase or decrease of more than 1.5 times when the strain was grown in co-culture with N. winogradskyi (Table 14 and 16). Of these 55 proteins 42 had an assigned function, while 13 were hypothetical proteins (Table 14 and 16). In total, the abundance of 24 proteins increased and 31 decreased in

! 74!

+ - - Figure 7. NH4 , NO2 and NO3 concentration in continuous cultures over time: Nitrosomonas sp. Is79 (A), Nitrosomonas sp. Is79 and N. winogradskyi (B and C) and the enrichment culture G5-7 (D and E).

! 75!

! !

5000

4000 A

3000 Ammonium Nitrite Concentration [uM] -

3 Nitrate 2000 or NO - 2 , NO +

4 1000 NH

0 0 100 200 300 400 500 600 Times (hours)

5000 5000

4000 B 4000 C

3000 3000 Concentration [uM] Concentration [uM] - - 3 3 2000 2000 or NO or NO - - 2 2 , NO , NO + +

4 1000 4 1000 NH NH

0 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Times (hours) Times (hours) ! !

5000 5000

4000 D 4000 E

3000 3000 Concentration [uM] Concentration [uM] - - 3 3 2000 2000 or NO or NO - - 2 2 , NO , NO + +

4 1000 4 1000 NH NH

0 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Times (hours) Times (hours) ! ! ! ! !

! 76! + - - Table 13. Steady state NH4 , NO2 and NO3 concentrations in the continuous cultures: Nitrosomonas sp. Is79 (Is79), Nitrosomonas sp. Is79 and N. winogradskyi (Is79+NOB) and the enrichment culture G5-7 (G5-7).

Is79 Is79+NOB G5-7 Run #1 Run #1 Run #2 Run #1 Run #2 + NH4 [µM] 115.95 2.54 6.35 1.88 0.92 - NO2 [µM] 4652.77 7.71 14.20 1.98 1.79 - NO3 [µM] n.d. 4667.06 4625.15 4541.53 4639.41 n.d.= not determined

! 77!

Figure 8. SDS-PAGE of whole cell extracts from the enrichment culture G5-7 (Lane 2 and 3), the co-culture Nitrosomonas sp. Is79 with N. winogradskyi (Lane 4 and 5) and the pure culture Nitrosomonas sp. Is79 (Lane 6) grown to steady state in continuous culture (Figure 7). Lane 1: molecular weight protein marker.

! 78!

!

!

! 79! Table 14. All Nitrosomonas sp. Is79 proteins that changed in abundance when in co-culture with N. winogradskyi (79 + NOB) compared to when grown as a pure culture. Log fold change values were converted to fold change values with negative values symbolizing decreased protein abundances and positive values symbolizing increased protein abundances. ! 79 + 79 + Sequence Locus p-value p-value Accession NOB NOB Confident covered Tag Description replicate replicate # replicate replicate Peptides (%) by Nit79A3 1 2 1 2 peptides gi|338803769 104 Succinyl-CoA ligase (ADP-forming) subunit beta -1.08 0.3687 -1.20 0.0103 25 55.7 gi|338803770 105 Succinyl-CoA synthetase, alpha subunit -1.33 0.061 -1.23 0.0974 17 58 gi|338803791 126 Phosphoenolpyruvate carboxylase -1.04 0.6857 1.04 0.5123 19 23.1 gi|338803807 142 6,7-dimethyl-8-ribityllumazine synthase -1.11 0.43 -1.37 0.112 9 65 gi|338803816 151 Isocitrate dehydrogenase, NADP-dependent -1.11 0.0604 -1.15 0.0016 20 41.9 gi|338803817 152 Cold-shock DNA-binding domain protein -1.65 0.1147 -1.5 0.0333 7 55.2 gi|338803826 161 Glycine hydroxymethyltransferase -1.20 0.021 -1.28 0.0131 25 40 gi|338803841 176 Ribose-5-phosphate isomerase A -1.03 0.7279 -1.24 0.0157 12 61.2 gi|338803889 224 Peptidoglycan-associated lipoprotein -1.18 0.0327 -1.29 0.0142 9 54.1 gi|338803890 225 Tol-pal system protein YbgF 1.39 0.2006 1.99 0.0291 4 14.6 gi|338803900 235 Phosphofructokinase -1.13 0.1187 -1.32 0.0195 36 79.2 gi|338803966 317 Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ Mal allergen -1.42 0.0383 -1.50 0.003 8 30.5 gi|338805151 336 Protein of unknown function DUF336 1.87 <0.0005 2.02 0.0005 12 64.2 gi|338804009 363 Hypothetical protein 2.61 0.5997 3.44 0.0112 3 23.1 gi|338804014 369 ATP synthase subunit b -1.43 0.2468 -1.46 0.0286 2 13.4 gi|338804015 370 ATP synthase subunit delta -1.5 0.0284 -1.73 0.0039 7 46.1 gi|338804016 371 ATP synthase subunit alpha -2.55 <0.0005 -3.20 <0.0005 59 55.9 gi|338804017 372 ATP synthase gamma chain -1.65 <0.0005 -1.91 <0.0005 20 53.1 gi|338804018 373 ATP synthase subunit beta -2.47 <0.0005 -2.84 <0.0005 78 80 gi|338804019 374 ATP synthase epsilon chain -1.68 0.0424 -1.66 0.074 5 17.9 gi|338804029 384 Cytochrome c oxidase, subunit II -1.46 0.0013 -1.36 0.0012 7 35.2 gi|338804049 407 Arginine biosynthesis bifunctional protein ArgJ -1.05 0.6532 -1.24 0.0305 11 30.6 gi|338804077 436 Hemolysin-type calcium-binding region 3.29 <0.0005 2.63 <0.0005 226 76.9 gi|338804081 440 Integration host factor subunit beta -1.83 0.0073 -1.25 0.2219 3 13.3 gi|338804082 441 Ribosomal protein S1 -1.13 0.2613 -1.24 0.0843 10 23.2 gi|338804096 455 Transketolase -1.20 0.005 -1.37 <0.0005 38 55.9 gi|338804097 456 Glyceraldehyde-3-phosphate dehydrogenase, type I -1.28 0.0038 1.52 <0.0005 27 66.3 gi|338804098 457 Phosphoglycerate kinase -1.64 <0.0005 -1.78 <0.0005 40 78.6 gi|338804100 459 Fructose-bisphosphate aldolase, class II, Calvin cycle subtype 1.35 0.0013 -1.43 0.0086 18 37.6 gi|338804162 524 Hypothetical protein 3.26 0.0053 2.88 0.0045 7 44.5 gi|338804197 560 Hypothetical protein 1.11 0.5194 1.36 0.0261 3 19.9 gi|338804209 572 Dihydroorotase, multifunctional complex type -1.53 <0.0005 -1.23 0.0381 9 34 gi|338804210 573 Peptidase M3A and M3B thimet/oligopeptidase F -1.05 0.7011 -1.17 0.0645 23 34.7 gi|338804216 579 PpiC-type peptidyl-prolyl cis-trans isomerase 1.45 0.0891 1.67 0.0017 14 38.3 gi|338804229 592 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase -1.26 0.1051 -1.45 0.044 3 8.5 gi|338804251 614 Phosphate binding protein 1.97 0.0247 1.30 0.1283 6 25

! 80! 79 + 79 + Sequence Locus p-value p-value Accession NOB NOB Confident covered Tag Description replicate replicate # replicate replicate Peptides (%) by Nit79A3 1 2 1 2 peptides gi|338804288 651 Short-chain dehydrogenase/reductase SDR -1.23 0.1803 -1.37 0.0111 11 46 gi|338804310 673 Aminotransferase class V -1.69 <0.0005 -2.03 <0.0005 45 67.4 gi|338804328 691 Ribosomal protein L13 -1.35 0.4749 1.02 0.9241 3 33.3 gi|338804387 754 Di-heme cytochrome c peroxidase 1.35 0.0062 1.89 0.0019 11 33.3 gi|338804428 795 Di-heme cytochrome c peroxidase 1.99 0.0006 2.99 0.000 22 52.4 gi|338804431 798 HflK protein -1.77 0.0212 -1.23 0.1091 5 15.6 gi|338804452 819 50S ribosomal protein L7/L12 -1.93 0.0011 -1.79 <0.0005 16 84.1 gi|338804466 833 Integration host factor subunit alpha -1.42 0.0667 -1.08 0.5607 6 46.1 gi|338804471 838 Hypothetical protein 2.57 0.0394 3.81 0.0033 8 45.8 gi|338804473 840 Homoserine dehydrogenase 1.29 0.1658 1.49 0.0272 14 41.2 gi|338804474 841 Threonine synthase -1.26 0.0229 -1.26 0.013 11 31.1 gi|338804480 847 Citrate synthase I 1.03 0.7097 -1.02 0.762 16 44.6 gi|338804512 884 Glutamyl-tRNA(Gln) amidotransferase subunit A -1.04 0.8212 -1.11 0.6293 5 16.8 gi|338804597 974 Hypothetical protein -2.70 0.0005 -1.67 0.0082 14 36.9 gi|338804618 997 Sulfate ABC transporter, periplasmic sulfate-binding protein 1.50 0.0024 1.72 0.0079 7 28.3 gi|338804625 1004 Phage tail sheath protein, putative 1.42 0.0397 1.26 0.0874 10 25.3 gi|338804637 1016 Hypothetical protein -1.22 0.0174 -1.22 0.0286 10 22.2 gi|338804691 1073 Peptidyl-prolyl cis-trans isomerase cyclophilin type 2.34 0.0084 1.56 0.0289 5 16.8 gi|338804754 1143 Uroporphyrinogen decarboxylase -1.18 0.0752 -1.38 0.0147 13 36.4 gi|338804785 1174 Extracellular solute-binding protein family 1 2.28 0.0008 2.17 0.0015 18 43.3 gi|338804826 1224 Glycine dehydrogenase (decarboxylating) subunit 1 -1.30 0.0108 -1.40 0.0011 34 58.2 gi|338804827 1225 Thiol peroxidase -1.75 0.0032 -1.52 0.0026 9 65.9 gi|338804828 1226 Glycine dehydrogenase (decarboxylating) subunit 2 -1.22 0.009 -1.41 0.0001 38 69.6 gi|338804855 1255 Ribulose bisphosphate carboxylase large chain -1.11 0.7311 -1.48 0.0459 6 13.7 gi|338804874 1275 Cytochrome c-type biogenesis protein CcmI -1.20 0.1886 -1.10 0.1486 6 18 gi|338804892 1299 Hypothetical protein -1.72 0.0683 -1.58 0.0088 3 45.8 gi|338804896 1303 Phosphoribulokinase/uridine kinase -1.36 0.0159 -1.19 0.1338 10 51.6 gi|338804901 1308 Peptidase C1A papain -1.26 0.0839 -1.44 0.0051 5 24 gi|338804920 1327 amidohydrolase 1.51 0.0152 1.29 0.1056 5 13.6 gi|338804952 1360 Peptidase M28 1.31 0.3735 1.40 0.0163 9 14.7 gi|338805124 1544 Glyoxalase/bleomycin resistance protein/dioxygenase -1.47 0.0402 -1.74 0.0038 10 55.1 gi|338805145 1567 Aldose 1-epimerase -1.14 0.4652 -1.23 0.0709 4 19.9 gi|338805167 1591 Polyribonucleotide nucleotidyltransferase -1.32 0.0003 -1.64 <0.0005 29 45.2 gi|338805185 1609 Hypothetical protein -1.47 <0.0005 -1.31 <0.0005 56 79.9 gi|338805200 1625 Ribonuclease T2 5.15 <0.0005 3.58 0.0003 17 48.4 gi|338805203 1628 Cytochrome P460 -1.48 0.0994 1.56 0.0046 20 60.4 gi|338805211 1637 Esterase/lipase/thioesterase family protein -1.42 0.035 1.07 0.3533 1 5.4 gi|338805260 1687 Succinyl-CoA ligase (ADP-forming) subunit beta -1.05 0.6457 -1.28 0.0068 13 43.2 gi|338805269 1697 Ribulose-phosphate 3-epimerase -1.52 0.0018 -1.77 0.0008 9 40.4 gi|338805276 1704 Glutamate-1-semialdehyde 2,1-aminomutase -1.12 0.208 -1.31 0.0108 22 64.3 gi|338805325 1753 Adenylosuccinate lyase -1.33 0.028 -1.10 0.5519 2 7.4 gi|338805327 1755 Chaperone protein dnaK -1.06 0.4096 -1.04 0.3578 42 60.1 gi|338805343 1774 UTP-glucose-1-phosphate uridylyltransferase -1.00 0.9827 -1.12 0.1341 13 44.8 gi|338805421 1860 Di-heme cytochrome c peroxidase 1.55 0.072 1.87 0.0127 8 28.6

! 81! 79 + 79 + Sequence Locus p-value p-value Accession NOB NOB Confident covered Tag Description replicate replicate # replicate replicate Peptides (%) by Nit79A3 1 2 1 2 peptides gi|338805427 1867 Hypothetical protein 1.75 0.0037 2.01 0.0006 10 37 gi|338805428 1868 Hypothetical protein 1.64 0.0051 1.70 0.0072 3 24.1 gi|338805431 1872 Hypothetical protein 1.08 0.9636 1.71 0.0383 1 6 gi|338805444 1888 Phosphoglucomutase/phosphomannomutase alpha/beta/alpha domain II 1.08 0.3297 -1.09 0.1493 24 43.2 gi|338805463 1907 2-oxo-acid dehydrogenase E1 subunit, homodimeric type 1.31 0.0074 1.17 0.1716 8 13.7 gi|338805468 1912 S-adenosylmethionine synthase -1.43 0.0024 -1.66 <0.0005 30 64.6 gi|338805471 1915 Type I phosphodiesterase/nucleotide pyrophosphatase 1.28 0.0016 1.06 0.3424 38 72.6 gi|338805484 1928 ATP-dependent Clp protease proteolytic subunit -1.20 0.0181 -1.46 0.0009 16 45.1 gi|338805487 1931 60 kDa chaperonin -1.42 <0.0005 -1.79 <0.0005 98 80.2 gi|338805488 1932 10 kDa chaperonin -1.59 0.0083 -1.57 0.001 7 63.5 gi|338805494 1938 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase -1.14 0.0202 1.01 0.6956 55 60.9 gi|338805505 1949 Dihydrolipoamide dehydrogenase -1.17 0.1656 -1.32 0.0289 13 38.7 gi|338805516 1960 Protease Do -1.44 0.0742 -1.42 0.0083 8 18.3 gi|338805517 1961 Sigma E regulatory protein, MucB/RseB 1.23 0.4818 1.26 0.1509 4 14.1 gi|338805541 1988 Manganese/iron superoxide dismutase -1.74 0.0021 -1.73 0.0008 30 80.3 gi|338805542 1989 ATP phosphoribosyltransferase -1.09 0.6051 -1.20 0.2444 10 55.6 gi|338805570 2021 Cysteine desulfurase, SufS subfamily -1.49 0.0006 -1.80 0.0012 8 21.4 gi|338805583 2034 Cytosol aminopeptidase -1.63 0.0382 -1.57 0.0076 4 10 gi|338805623 2076 NusA antitermination factor -1.22 0.0363 -1.36 0.0035 13 40.6 gi|338805641 2094 Enolase -1.16 0.1405 -1.20 0.0073 16 48.7 gi|338805661 2114 Fumarate hydratase class II -1.30 0.2717 -1.24 0.23 6 15.8 gi|338805707 2165 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase 1.58 0.0642 1.20 0.2804 4 15.8 Deoxyribose-phosphate aldolase/phospho-2-dehydro-3-deoxyheptonate gi|338805743 2202 -1.16 0.2108 -1.30 0.0085 16 46.7 aldolase gi|338805786 2245 Dihydroxy-acid dehydratase -1.19 0.0033 -1.25 0.0009 16 29.1 gi|338805805 2265 Hypothetical protein -1.03 0.7689 1.10 0.2978 19 48.9 gi|338805859 2329 PAS sensor protein -1.19 0.2696 -1.18 0.3279 7 59.3 gi|338805865 2335 Nitrite reductase, copper-containing 2.40 0.0032 3.54 0.0001 51 55.6 gi|338805889 2359 Hypothetical protein 1.85 0.0177 2.49 0.007 6 38.3 gi|338805895 2365 Glutamine synthetase, type I -1.40 0.0018 -1.54 <0.0005 24 51 gi|338805974 2444 Hypothetical protein 2.21 <0.0005 1.84 0.0002 32 47.7 gi|338806003 2473 FimV N-terminal domain-containing protein -1.28 0.3506 -1.47 0.0178 5 7.8 gi|338806009 2479 Rubrerythrin -1.80 0.0021 -1.51 0.0001 30 74.8 gi|338806036 2508 6-phosphogluconate dehydrogenase, decarboxylating -2.36 0.001 -2.18 0.0019 8 30.3 gi|338806043 2515 Nitrogen regulatory protein P-II -2.38 0.0001 -2.11 <0.0005 4 33 gi|338806059 2533 Succinate dehydrogenase, flavoprotein subunit -1.59 0.0081 -1.48 0.0181 1 2.4 gi|338806101 2576 Hypothetical protein -1.15 0.2516 -1.12 0.1478 14 47.3 gi|338806102 2577 Hypothetical protein 1.06 0.7887 -1.02 0.7775 7 41.8 gi|338806154 2629 OmpA/MotB domain protein -1.54 0.0074 -1.51 0.0026 17 58.5 gi|338806237 2723 Protein of unknown function DUF1458 -1.33 0.0544 -1.45 0.0154 5 58.8 gi|338806270 2758 Hypothetical protein 1.52 0.0401 1.10 0.5504 5 32.6 gi|338806285 2776 Lipase class 3 2.18 0.0002 2.13 <0.0005 19 58 gi|338806299 2790 Delta-aminolevulinic acid dehydratase -1.19 0.2489 -1.22 0.0088 12 36.4 gi|338806304 2795 DNA-directed RNA polymerase subunit alpha -1.22 0.0766 -1.19 0.0258 16 49.1

! 82! 79 + 79 + Sequence Locus p-value p-value Accession NOB NOB Confident covered Tag Description replicate replicate # replicate replicate Peptides (%) by Nit79A3 1 2 1 2 peptides gi|338806311 2802 Ribosomal protein L15 1.32 0.025 1.02 0.7884 3 21 gi|338806325 2816 Ribosomal protein L22 -1.01 0.9714 1.06 0.3411 4 21.7 gi|338806329 2820 Ribosomal protein L4/L1e -1.03 0.7932 1.16 0.1291 15 62.1 gi|338806332 2823 Translation elongation factor Tu 1.04 0.5381 1.12 0.0036 46 77 gi|338806340 2831 DNA gyrase, A subunit 1.70 0.0239 1.08 0.7953 5 6.2 gi|338806350 2841 Hypothetical protein -1.31 0.0339 -1.46 0.0046 14 69.7 gi|338806367 2858 Domain of unknown function DUF1993-containing protein -1.37 0.0931 -1.16 0.3965 8 37.1 gi|338806370 2861 Tetratricopeptide TPR_2 repeat-containing protein 1.56 0.041 1.45 0.0032 4 6.9 gi|338806391 2882 Hypothetical protein -1.91 0.0207 -2.04 0.005 8 42 gi|338806393 2884 Ammonia monooxygenase, subunit B 1.23 0.0063 1.42 <0.0005 63 63.6 gi|338806394 2885 ammonia monooxygenase, subunit A 1.20 0.2439 1.46 0.0434 6 15.3 gi|338806404 2895 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase -1.18 0.1505 -1.20 0.0206 13 38.1 gi|338806408 2899 Short-chain dehydrogenase/reductase SDR -1.21 0.1356 -1.13 0.379 6 16.8 gi|338806421 2912 Hypothetical protein -1.17 0.0483 -1.05 0.6657 11 67.1 gi|338806437 2928 NADH-quinone oxidoreductase, F subunit -1.09 0.409 -1.17 0.0363 10 27.8 gi|338806444 2935 Triosephosphate isomerase -1.15 0.1569 -1.26 0.0353 15 55.4 gi|338806449 2940 Cytochrome c-554 -1.16 0.1467 4.92 0.0002 8 28.5 gi|338806451 2942 Hydroxylamine dehydrogenase -1.43 <0.0005 1.11 0.0977 61 51.7 gi|338806452 2943 Hypothetical protein -1.77 0.0219 -1.98 0.1759 3 12.5 gi|338806490 2984 Hypothetical protein 1.57 0.0078 1.44 0.065 4 26.4 gi|338806500 2995 Glucose sorbosone dehydrogenase 1.43 0.0916 1.46 0.0791 10 35.6 gi|338806518 3014 Alpha-glucan phosphorylase -1.18 0.2251 -1.24 0.002 16 25.3 gi|338806520 3017 Hypothetical protein -1.18 0.0002 -1.32 <0.0005 54 47.8 gi|338806539 3041 Glucose-1-phosphate adenylyltransferase -1.22 0.0113 -1.26 0.0207 11 27.6 gi|338806549 3051 Redoxin domain protein -1.17 0.2817 -1.01 0.8859 6 39.2 gi|338806550 3052 Ferritin Dps family protein 1.41 0.2682 1.63 0.0284 5 52.6 gi|338806603 3107 Hypothetical protein 1.96 0.0001 1.53 0.0001 39 67.6 gi|338806604 3108 Hypothetical protein 2.26 0.0004 1.38 0.0035 34 82.3 gi|338806639 3143 Acyl carrier protein -1.53 0.0111 -1.56 0.129 6 48.7 gi|338806646 3150 Ankyrin 1.97 0.0024 1.75 0.0096 15 56.5 gi|338806650 3154 Hypothetical protein -1.52 0.001 -1.65 <0.0005 34 62.8 gi|338806662 3169 Bacterioferritin -1.25 0.4714 2.47 0.0088 2 23.4 gi|338806665 3172 Single-strand binding protein -1.39 0.2088 -1.38 0.0163 5 32.9 gi|338806671 3178 Porin Gram-negative type -1.24 0.023 1.05 0.5345 20 52.4 gi|338806725 3233 Phosphoenolpyruvate synthase 1.21 0.0345 1.07 0.5186 8 14.3 gi|338806727 3235 Ribulose bisphosphate carboxylase large chain -1.19 0.0023 -1.23 <0.0005 90 66 gi|338806728 3236 Ribulose bisphosphate carboxylase small chain -1.28 0.0096 -1.27 <0.0005 44 95.8 gi|338806799 3310 Alkaline phosphatase -1.23 0.0271 -1.23 0.094 6 14.9 gi|338806806 3317 Glu/Leu/Phe/Val dehydrogenase 1.24 0.0011 1.08 0.1589 28 69.6 gi|338806819 3332 Protein of unknown function DUF488 -3.74 0.1529 -3.79 0.0526 5 40 gi|338806861 3379 Alanine dehydrogenase/PNT domain protein -1.35 0.001 -1.51 <0.0005 25 54.9 gi|338806863 3381 FAD-dependent pyridine nucleotide-disulfide oxidoreductase 1.32 0.0015 1.18 0.0009 25 28.7 gi|338806881 3399 Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ Mal allergen -1.42 0.0057 -1.33 0.0202 7 36.5 gi|338806955 3475 1-deoxy-D-xylulose 5-phosphate reductoisomerase -1.03 0.8915 -1.19 0.1053 2 10.6

! 83! 79 + 79 + Sequence Locus p-value p-value Accession NOB NOB Confident covered Tag Description replicate replicate # replicate replicate Peptides (%) by Nit79A3 1 2 1 2 peptides gi|338806966 3487 Hypothetical protein -1.84 0.0246 -1.74 0.0377 8 37.3 gi|338806969 3490 Peptidase M4 thermolysin -1.42 <0.0005 -1.36 <0.0005 112 79.3 gi|338807030 3552 Copper resistance protein CopC 2.10 0.0071 1.91 0.0442 5 43.3 !

! 84! Table 15. All Nitrosomonas sp. Is79 proteins that changed in abundance when part of the enrichment culture G5-7 (G5-7) compared to when grown as a pure culture. Log fold change values were converted to fold change values with negative values symbolizing decreased protein abundances and positive values symbolizing increased protein abundances. ! Sequence Locus G5-7 p-value G5-7 p-value Accession Confident covered Tag Description replicate replicate replicate replicate # Peptides (%) by Nit79A3 1 1 2 2 peptides gi|338803669 2 DNA polymerase III, beta subunit -1.62 <0.0005 -1.52 0.0003 11 32.1 gi|338803768 103 HpcH/HpaI aldolase 1.44 0.0005 1.36 0.0007 13 46.2 gi|338803769 104 Succinyl-CoA ligase (ADP-forming) subunit beta -1.66 <0.0005 -1.67 <0.0005 18 50.1 gi|338803770 105 Succinyl-CoA synthetase, alpha subunit -1.55 0.0104 -1.73 0.0222 11 42 gi|338803816 151 Isocitrate dehydrogenase, NADP-dependent -1.33 0.0015 -1.38 <0.0005 20 43.4 gi|338803817 152 Cold-shock DNA-binding domain protein -2.26 0.0442 -2.34 0.0122 5 55.2 gi|338803826 161 Glycine hydroxymethyltransferase -1.60 <0.0005 -1.69 <0.0005 27 46.5 gi|338803831 166 DAHP synthetase I/KDSA -1.40 0.0087 -1.27 0.094 6 25.8 gi|338803841 176 Ribose-5-phosphate isomerase A -1.46 0.047 -1.46 0.0405 11 42.9 gi|338803889 224 Peptidoglycan-associated lipoprotein -1.42 0.1694 -1.24 0.243 6 43 gi|338803900 235 Phosphofructokinase -1.48 <0.0005 -1.7 <0.0005 34 61.8 gi|338803966 317 Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ Mal allergen -1.37 0.0614 -1.55 0.026 5 23.5 gi|338804015 370 ATP synthase subunit delta -1.92 0.0272 -1.61 0.0238 6 35.4 gi|338804016 371 ATP synthase subunit alpha -5.35 <0.0005 -5.01 <0.0005 47 56.1 gi|338804017 372 ATP synthase gamma chain -2.05 <0.0005 -2.17 <0.0005 18 51.4 gi|338804018 373 ATP synthase subunit beta -4.12 <0.0005 -3.98 <0.0005 65 78.6 gi|338804077 436 Hemolysin-type calcium-binding region 1.43 0.0621 1.91 0.0066 182 71.5 gi|338804081 440 Integration host factor subunit beta 1.08 0.4539 -1.23 0.0706 3 20.4 gi|338804082 441 Ribosomal protein S1 -1.05 0.668 1.00 0.9793 11 25.6 gi|338804096 455 Transketolase -1.57 <0.0005 -1.83 <0.0005 31 46.7 gi|338804097 456 Glyceraldehyde-3-phosphate dehydrogenase, type I 1.46 0.0003 1.26 0.0068 32 72.3 gi|338804098 457 Phosphoglycerate kinase -3.14 <0.0005 -2.89 <0.0005 38 79.3 gi|338804099 458 Pyruvate kinase -1.40 0.0425 -1.33 0.0825 13 35.6 gi|338804100 459 Fructose-bisphosphate aldolase, class II, Calvin cycle subtype 1.35 0.0283 -1.01 0.8968 17 35.6 gi|338804106 466 Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ Mal allergen -1.53 0.0249 -1.58 0.068 2 5.9 gi|338804162 524 Hypothetical protein 2.58 0.0409 1.52 0.1348 6 43.9 gi|338804189 552 Acylneuraminate cytidylyltransferase -1.68 0.158 -1.40 0.0325 5 22.1 gi|338804197 560 Hypothetical protein 1.44 0.0513 1.56 0.0395 2 15.8 gi|338804199 562 50S ribosomal protein L19 -1.01 0.9245 1.16 0.0564 6 51.1 gi|338804210 573 Peptidase M3A and M3B thimet/oligopeptidase F -1.16 0.275 -1.26 0.0296 13 25.7 gi|338804216 579 PpiC-type peptidyl-prolyl cis-trans isomerase 1.72 0.0046 1.94 0.0011 13 37.5 gi|338804249 612 Nicotinate-nucleotide pyrophosphorylase -1.19 0.6412 -1.12 0.7227 1 5.9

! 85! Sequence Locus G5-7 p-value G5-7 p-value Accession Confident covered Tag Description replicate replicate replicate replicate # Peptides (%) by Nit79A3 1 1 2 2 peptides gi|338804251 614 Phosphate binding protein 1.65 0.0228 1.48 0.0264 8 27.1 gi|338804278 641 Carboxyl-terminal protease 1.08 0.6116 1.06 0.7388 9 25 gi|338804286 649 Histone family protein DNA-binding protein 3.96 0.0448 2.07 0.1322 7 43.3 gi|338804287 650 PpiC-type peptidyl-prolyl cis-trans isomerase -1.72 0.0366 -1.21 0.0397 4 10.8 gi|338804288 651 Short-chain dehydrogenase/reductase SDR -1.33 0.2167 -1.31 0.048 10 37.2 gi|338804310 673 Aminotransferase class V -2.57 <0.0005 -2.67 <0.0005 36 61 gi|338804315 678 dTDP-4-dehydrorhamnose 3,5-epimerase -2.14 0.0742 -1.69 0.0345 2 13.2 gi|338804387 754 Di-heme cytochrome c peroxidase 1.39 0.0907 1.43 0.077 10 38.5 gi|338804419 786 Acetolactate synthase, small subunit -1.17 0.0817 -1.28 0.0417 7 47.2 gi|338804420 787 Ketol-acid reductoisomerase 1.41 0.0208 1.47 0.0008 14 43.2 gi|338804428 795 Di-heme cytochrome c peroxidase 3.28 0.0019 2.32 0.0144 36 57 gi|338804452 819 50S ribosomal protein L7/L12 -2.32 0.0046 -1.86 0.0013 13 84.1 gi|338804453 820 DNA-directed RNA polymerase subunit beta 1.39 0.0028 1.32 0.0022 24 24.2 gi|338804454 821 DNA-directed RNA polymerase subunit beta' 1.20 0.035 1.22 0.009 19 19.7 gi|338804466 833 Integration host factor subunit alpha 1.05 0.6327 -1.31 0.0666 7 46.1 gi|338804471 838 Hypothetical protein 3.59 0.0513 2.96 0.0055 8 35.6 gi|338804473 840 Homoserine dehydrogenase 1.37 0.116 1.26 0.2274 8 28.4 gi|338804474 841 Threonine synthase -1.65 0.0001 -1.48 0.0015 13 39.4 gi|338804480 847 Citrate synthase I 1.01 0.8638 -1.17 0.0045 17 43.2 gi|338804597 974 Hypothetical protein -1.25 0.2411 -1.68 0.0019 10 36.9 gi|338804618 997 Sulfate ABC transporter, periplasmic sulfate-binding protein 2.09 0.0037 1.78 0.0025 11 32.8 gi|338804625 1004 Phage tail sheath protein, putative 2.07 <0.0005 1.56 0.0001 26 62.6 gi|338804637 1016 Hypothetical protein 1.10 0.3601 -1.05 0.5604 14 31.3 gi|338804639 1018 Hypothetical protein 1.72 <0.0005 1.61 0.0008 13 38.7 gi|338804646 1025 Peptidoglycan-binding domain-containing protein 2.79 0.0074 2.73 0.0013 7 35.7 gi|338804649 1028 Hypothetical protein 1.64 0.0217 1.67 0.0154 8 23.9 gi|338804691 1073 Peptidyl-prolyl cis-trans isomerase cyclophilin type 1.54 0.0369 1.66 0.0501 5 14.1 gi|338804754 1143 Uroporphyrinogen decarboxylase -1.59 0.0182 -1.54 0.0205 10 35.6 gi|338804785 1174 Extracellular solute-binding protein family 1 2.22 <0.0005 1.85 <0.0005 19 59 gi|338804788 1177 Thioredoxin -1.16 0.3164 -1.20 0.0474 8 44.4 gi|338804826 1224 Glycine dehydrogenase (decarboxylating) subunit 1 -1.99 <0.0005 -2.26 <0.0005 25 58.2 gi|338804827 1225 Thiol peroxidase -2.19 0.0041 -1.95 0.0001 9 70.1 gi|338804828 1226 Glycine dehydrogenase (decarboxylating) subunit 2 -1.75 <0.0005 -1.93 <0.0005 38 71.4 gi|338804892 1299 Hypothetical protein -2.25 0.0006 -1.91 0.0001 5 34.2 gi|338804896 1303 Phosphoribulokinase/uridine kinase 1.34 0.0271 1.14 0.1528 11 53.3 gi|338804901 1308 Peptidase C1A papain -1.36 0.0547 -1.42 0.0099 4 15.5 gi|338805124 1544 Glyoxalase/bleomycin resistance protein/dioxygenase -2.07 0.0286 -1.73 0.075 5 39.4 gi|338805167 1591 Polyribonucleotide nucleotidyltransferase -1.52 <0.0005 -1.57 <0.0005 28 43.6 gi|338805185 1609 Hypothetical protein -1.49 <0.0005 -1.89 <0.0005 52 79.1 gi|338805200 1625 Ribonuclease T2 2.93 0.0008 2.83 0.002 11 38.2

! 86! Sequence Locus G5-7 p-value G5-7 p-value Accession Confident covered Tag Description replicate replicate replicate replicate # Peptides (%) by Nit79A3 1 1 2 2 peptides gi|338805203 1628 Cytochrome P460 2.96 0.0011 2.46 0.0004 24 70.1 gi|338805267 1695 Anthranilate synthase component I -1.60 0.0283 -1.48 0.0399 3 12.9 gi|338805269 1697 Ribulose-phosphate 3-epimerase -2.97 0.0005 -2.98 0.0003 9 40 gi|338805276 1704 Glutamate-1-semialdehyde 2,1-aminomutase -1.50 0.0029 -1.57 0.0012 18 64.6 gi|338805327 1755 Chaperone protein dnaK -1.05 0.6289 -1.13 0.2013 45 66.9 gi|338805342 1773 Hypothetical protein -1.08 0.8515 1.48 0.1858 6 66.1 gi|338805343 1774 UTP-glucose-1-phosphate uridylyltransferase -1.27 0.0317 -1.23 0.0405 11 50.8 gi|338805351 1783 Protein of unknown function UPF0001 1.43 0.1033 1.20 0.1535 3 7.5 gi|338805421 1860 Di-heme cytochrome c peroxidase 1.98 0.0116 1.61 0.078 20 51.1 gi|338805427 1867 Hypothetical protein 3.14 0.0004 2.13 0.0041 14 46.1 gi|338805428 1868 Hypothetical protein 2.17 0.0033 1.54 0.0502 5 32.1 gi|338805431 1872 Hypothetical protein 1.54 0.0292 1.48 0.0671 2 12.4 gi|338805432 1873 Putative transmembrane protein 1.22 0.2989 1.56 0.034 3 21.3 gi|338805442 1886 Branched-chain amino acid aminotransferase -1.22 0.0598 -1.29 0.0299 9 39.2 gi|338805444 1888 Phosphoglucomutase/phosphomannomutase alpha/beta/alpha domain II -1.27 0.0482 -1.19 0.1005 13 29.5 gi|338805467 1911 Adenosylhomocysteinase 1.06 0.4107 -1.40 <0.0005 45 57.9 gi|338805468 1912 S-adenosylmethionine synthase -2.19 0.0001 -2.42 <0.0005 25 57.1 gi|338805484 1928 ATP-dependent Clp protease proteolytic subunit -1.73 0.0148 -1.69 0.0116 11 37.6 gi|338805487 1931 60 kDa chaperonin -1.82 <0.0005 -2.15 <0.0005 90 73.4 gi|338805488 1932 10 kDa chaperonin -1.85 0.0149 -2.11 0.005 6 53.1 gi|338805494 1938 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase 1.22 0.004 -1.15 0.0408 56 64.3 gi|338805498 1942 Glutaredoxin-like protein -1.98 0.0633 -2.00 0.0214 6 57.8 gi|338805504 1948 Hypothetical protein 1.70 0.0183 1.28 0.3132 5 35.4 gi|338805505 1949 Dihydrolipoamide dehydrogenase -1.67 0.0213 -1.55 0.0298 12 35.4 gi|338805517 1961 Sigma E regulatory protein, MucB/RseB 1.72 0.0075 1.85 0.0016 6 19.5 gi|338805541 1988 Manganese/iron superoxide dismutase -2.29 0.0001 -2.73 0.0001 33 80.3 gi|338805542 1989 ATP phosphoribosyltransferase -1.53 0.0142 -1.42 0.0078 12 59.4 gi|338805543 1990 Histidinol dehydrogenase 1.26 0.0168 1.18 0.1182 6 16.8 gi|338805570 2021 Cysteine desulfurase, SufS subfamily -2.21 0.0099 -2.18 0.0159 5 12.4 gi|338805605 2056 Coproporphyrinogen-III oxidase, aerobic -1.35 <0.0005 -1.24 0.0096 13 53.7 gi|338805623 2076 NusA antitermination factor -1.16 0.2063 -1.26 0.0314 11 26.7 gi|338805641 2094 Enolase -1.39 0.012 -1.65 <0.0005 20 48.2 gi|338805670 2124 Orn/DAP/Arg decarboxylase 2 -1.68 0.0805 -1.37 0.0366 2 6.4 gi|338805689 2147 Hypothetical protein -1.40 0.0165 -1.42 0.002 9 30.4 gi|338805705 2163 Protein of unknown function -1.74 0.031 -1.09 0.4484 1 6.1 gi|338805750 2209 Phosphoribosylamine--glycine ligase -1.51 0.0436 -1.18 0.1143 6 20.5 gi|338805779 2238 Aminotransferase class IV -1.59 0.0324 -1.56 0.0123 10 45.5 gi|338805786 2245 Dihydroxy-acid dehydratase -1.48 0.1638 -1.45 <0.0005 13 24.6 gi|338805805 2265 Hypothetical protein 1.79 <0.0005 1.41 0.0019 21 55.5 gi|338805859 2329 PAS sensor protein -1.36 0.0155 -1.40 0.02 7 54.7

! 87! Sequence Locus G5-7 p-value G5-7 p-value Accession Confident covered Tag Description replicate replicate replicate replicate # Peptides (%) by Nit79A3 1 1 2 2 peptides gi|338805860 2330 Polyketide cyclase/dehydrase 1.28 0.1215 1.35 0.0388 6 28.9 gi|338805865 2335 Nitrite reductase, copper-containing 4.34 <0.0005 3.00 <0.0005 80 66 gi|338805895 2365 Glutamine synthetase, type I -2.09 <0.0005 -2.21 <0.0005 23 43.1 gi|338805974 2444 Hypothetical protein 2.10 <0.0005 1.59 0.001 31 51.3 gi|338805991 2461 Peptidyl-prolyl cis-trans isomerase cyclophilin type 2.61 0.0408 1.78 0.1317 3 17.3 gi|338805993 2463 O-succinylhomoserine sulfhydrylase -1.39 0.008 -1.47 0.0079 11 29.9 gi|338806000 2470 Tryptophan synthase beta chain -1.03 0.8366 1.22 0.0861 5 10.5 gi|338806009 2479 Rubrerythrin -2.00 0.0002 -2.33 <0.0005 30 80.6 gi|338806010 2480 Hypothetical protein -1.01 0.9823 1.61 0.0776 4 9.7 gi|338806028 2499 Hypothetical protein -1.08 0.5306 -1.02 0.873 15 32.7 gi|338806036 2508 6-phosphogluconate dehydrogenase, decarboxylating -1.58 0.0378 -1.66 0.0273 7 33.2 gi|338806037 2509 Hypothetical protein 1.54 0.0146 1.73 0.0698 3 22.5 gi|338806043 2515 Nitrogen regulatory protein P-II -1.52 0.0178 -1.52 0.0098 4 32 gi|338806101 2576 Hypothetical protein -1.47 0.0011 -1.77 0.0047 9 42.6 gi|338806154 2629 OmpA/MotB domain protein -1.64 0.0067 -1.63 0.0035 16 59 gi|338806237 2723 Protein of unknown function -1.16 0.5371 -1.31 0.2893 4 48.5 gi|338806285 2776 Lipase class 3 3.12 <0.0005 2.53 0.0001 22 65.6 gi|338806315 2806 Ribosomal protein L6 1.22 0.2876 1.35 0.0415 8 42.5 gi|338806325 2816 Ribosomal protein L22 -1.18 0.3137 -1.05 0.5848 6 35.6 gi|338806328 2819 Ribosomal protein L25/L23 -1.31 0.0335 -1.03 0.781 2 21.6 gi|338806332 2823 Translation elongation factor Tu 2.14 <0.0005 1.63 <0.0005 65 78.5 gi|338806333 2824 Translation elongation factor G 1.18 0.1849 1.18 0.0404 24 47 gi|338806339 2830 Acetylornithine/succinyldiaminopimelate aminotransferase -1.30 0.0656 -1.22 0.0474 9 26.7 gi|338806350 2841 Hypothetical protein -1.69 0.002 -1.79 0.0022 11 76.8 gi|338806391 2882 Hypothetical protein -2.78 0.0027 -2.64 0.0051 5 42 gi|338806393 2884 Ammonia monooxygenase, subunit B 1.99 <0.0005 1.7 <0.0005 82 63.8 gi|338806394 2885 Ammonia monooxygenase, subunit A 2.04 0.0115 1.93 0.0235 5 12.8 gi|338806404 2895 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase -1.10 0.4142 -1.55 0.005 16 42.1 gi|338806412 2903 Hypothetical protein -1.56 0.0228 -1.38 0.2875 4 33.7 gi|338806428 2919 Chaperone protein htpG -1.55 0.0426 -1.21 0.597 2 4.9 gi|338806438 2929 NADH-quinone oxidoreductase, E subunit -1.62 0.0271 -1.29 0.1682 2 17.7 gi|338806440 2931 NAD(P)H-quinone oxidoreductase subunit J -1.17 0.1852 -1.53 0.0117 5 36.9 gi|338806444 2935 Triosephosphate isomerase -1.61 0.0037 -1.79 0.005 14 43.8 gi|338806449 2940 Cytochrome c-554 7.13 <0.0005 3.58 0.0008 8 28.5 gi|338806451 2942 Hydroxylamine dehydrogenase 1.74 <0.0005 1.28 0.0009 83 57.9 gi|338806500 2995 Glucose sorbosone dehydrogenase 1.34 0.0466 1.47 0.0114 7 28.1 gi|338806518 3014 Alpha-glucan phosphorylase -2.03 0.0098 -1.79 0.0077 10 13.4 gi|338806540 3042 Glycoside hydrolase family 57 2.76 0.0228 2.03 0.1196 3 9 gi|338806520 3107 Hypothetical protein -1.17 0.003 -1.35 <0.0005 54 47.7 gi|338806603 3107 Hypothetical protein 1.28 0.0281 1.44 0.0017 32 61.3

! 88! Sequence Locus G5-7 p-value G5-7 p-value Accession Confident covered Tag Description replicate replicate replicate replicate # Peptides (%) by Nit79A3 1 1 2 2 peptides gi|338806604 3108 Hypothetical protein 1.47 0.0411 1.04 0.7726 27 82.3 gi|338806640 3144 3-oxoacyl-(acyl-carrier-protein) synthase 2 -1.31 0.040 -1.36 0.0189 9 26.2 gi|338806646 3150 Ankyrin 1.29 0.0135 1.34 0.0031 11 43 gi|338806650 3154 Hypothetical protein -2.00 0.0015 -2.54 0.0001 24 56.6 gi|338806662 3169 Bacterioferritin 2.66 0.0404 2.15 0.0922 10 29.2 gi|338806665 3172 Single-strand binding protein -1.67 0.0565 -1.87 0.0064 4 20.1 gi|338806671 3178 Porin Gram-negative type -1.38 0.0151 -1.31 0.0099 18 47.4 gi|338806702 3210 Protein of unknown function -1.35 0.2064 -1.38 0.0005 9 31 gi|338806725 3233 Phosphoenolpyruvate synthase 1.29 0.026 1.12 0.114 13 25.2 gi|338806727 3235 Ribulose bisphosphate carboxylase large chain -1.15 0.0073 -1.58 <0.0005 94 67.4 gi|338806728 3236 Ribulose bisphosphate carboxylase small chain -1.17 0.0167 -1.54 <0.0005 47 95.8 gi|338806797 3308 Hypothetical protein -1.09 0.6077 -1.19 0.1595 4 15.1 gi|338806799 3310 Alkaline phosphatase -1.31 0.0374 -1.37 0.0414 4 8.2 gi|338806806 3317 Glu/Leu/Phe/Val dehydrogenase -1.52 0.0002 -1.5 <0.0005 24 57.3 gi|338806861 3379 Alanine dehydrogenase/PNT domain protein -2.15 <0.0005 -2.01 <0.0005 17 44.5 gi|338806881 3399 Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ Mal allergen -1.28 0.0127 -1.24 0.0102 10 59.8 gi|338806918 3437 Heat shock protein Hsp20 1.58 0.0449 -1.05 0.5554 9 50.3 gi|338806965 3486 Di-heme cytochrome c peroxidase 2.71 0.0519 3.12 0.0453 1 31.1 gi|338806966 3487 Hypothetical protein -2.31 <0.0005 -2.69 0.0015 7 27.6 gi|338806969 3490 Peptidase M4 thermolysin -1.29 0.0003 -1.73 <0.0005 118 79.6 gi|338806981 3502 Peptidylprolyl isomerase FKBP-type 1.36 0.2482 1.42 0.1451 4 38.3 !

! 89! Table 16. Nitrosomonas sp. Is79 proteins changed in abundance by at least 1.5 times in co-culture with N. winogradskyi (Is79 + NOB) and in the enrichment culture G5-7 (G5-7) (p<0.01). To determine the effect of the heterotrophic community of G5-7 the effect of N. winogradskyi on the proteome of Nitrosomonas sp. Is79 was subtracted from the effect of the overall community present in the enrichment culture G5-7.

The influence of Heterotrophic Locus N. G5-7 community Accession # Tag: Description winogradskyi (Rep1/Rep2) of G5-7 Nit79A3 (Rep1/Rep2) (Rep1/Rep2) Energy Generation and Nitrogen Metabolism

gi|338804015 370 ATP synthase F1 complex subunit delta -1.50 / -1.73 - - gi|338804016 371 ATP synthase F1 complex subunit alpha -2.55 / -3.20 -5.35 / -5.01 - gi|338804017 372 ATP synthase F1 complex subunit gamma -1.65 / -1.91 -2.05 / -2.17 - gi|338804018 373 ATP synthase F1 complex subunit beta -2.47 / -2.84 -4.12 / -3.98 - gi|338805865 2335 Nitrite reductase, copper-containing (NirK) 2.40 / 3.54 4.34 / 3.00 - gi|338806391 2882 Hypothetical protein: ORF5 -1.91 / -2.04 -2.78 / -2.64 - gi|338806393 2884 Ammonia monooxygenase, subunit B - 1.99 / 1.70 1.99 / 1.70 gi|338806394 2885 Ammonia monooxygenase, subunit A - 2.04 / 1.93 2.04 / 1.93 gi|338806449 2940 Cytochrome c-554 - 7.13 / 3.58 7.13 / 3.58 gi|338806451 2942 Hydroxylamine dehydrogenase - 1.74 / 1.28 1.74 / 1.28 gi|338805203 1628 Cytochrome P460 - 2.96 / 2.46 2.96 / 2.46 Oxidative Stress Response gi|338803966 317 Alkyl hydroperoxide reductase/ Thiol specific antioxidant -1.42 / -1.50 - - gi|338804387 754 Di-heme cytochrome c peroxidase 1.35 / 1.89 - - gi|338804428 795 Di-heme cytochrome c peroxidase 1.99 / 2.99 3.28 / 2.32 - gi|338805421 1860 Di-heme cytochrome c peroxidase 1.55 / 1.87 - - gi|338805487 1931 60 kDa chaperonin (GroEL) -1.42 / -1.79 -1.82 / -2.15 - gi|338805488 1932 10 kDa chaperonin (GroES) -1.59 / -1.57 -1.85 / -2.11 - gi|338804827 1225 Thiol peroxidase -1.75 / -1.52 -2.19 / -1.95 - gi|338805541 1988 Manganese/iron superoxide dismutase -1.74 / -1.73 -2.29 / -2.73 -

! 90! The influence of Heterotrophic Locus N. G5-7 community Accession # Tag: Description winogradskyi (Rep1/Rep2) of G5-7 Nit79A3 (Rep1/Rep2) (Rep1/Rep2) gi|338805570 2021 Cysteine desulfurase, SufS subfamily -1.49 / -1.80 -2.21 / -2.18 - gi|338806009 2479 Rubrerythrin -1.80 / -1.51 -2.00 / -2.33 - gi|338806646 3150 Ankyrin 1.97 / 1.75 - - gi|338805517 1961 Sigma E regulatory protein, MucB/RseB - 1.72 / 1.85 1.72 / 1.85 Carbon Metabolism gi|338804098 457 Phosphoglycerate kinase -1.64 /-1.78 -3.14 / -2.89 - gi|338805124 1544 Glyoxalase/bleomycin resistance protein/dioxygenase -1.47 / -1.74 - - gi|338805269 1697 Ribulose-phosphate 3-epimerase -1.52 /-1.77 -2.97 / -2.98 - gi|338806036 2508 6-phosphogluconate dehydrogenase -2.36 /-2.18 - - gi|338806059 2533 Succinate dehydrogenase, flavoprotein subunit -1.59 /-1.48 - - gi|338803769 104 Succinyl-CoA ligase (ADP-forming) subunit beta - -1.66 / -1.67 -1.66 / -1.67 gi|338803900 235 Phosphofructokinase - -1.48 / -1.70 -1.48 / -1.70 gi|338804096 455 Transketolase - -1.57 / -1.83 -1.57 / -1.83 gi|338805641 2094 Enolase - -1.39 / -1.65 -1.39 / -1.65 gi|338806444 2935 Triosephosphate isomerase - -1.61 / -1.79 -1.61 / -1.79 gi|338806728 3236 Ribulose bisphosphate carboxylase small chain - -1.17 / -1.54 -1.17 / -1.54 gi|338806727 3235 Ribulose bisphosphate carboxylase large chain - -1.15 / -1.58 -1.15 / -1.58 Nutrient Binding gi|338804077 436 Hemolysin-type calcium-binding region 3.29 / 2.63 1.43 / 1.91 - Sulfate ABC transporter, periplasmic sulfate-binding gi|338804618 997 1.50 / 1.72 2.09 / 1.78 - protein gi|338804785 1174 Extracellular solute-binding protein family 1 2.28 / 2.17 2.22 / 1.85 - gi|338806154 2629 OmpA/MotB domain protein -1.54 / -1.51 -1.64 / -1.63 - gi|338807030 3552 Copper resistance protein CopC 2.10 / 1.91 - - Macromolecule Degradation gi|338805167 1591 Polyribonucleotide nucleotidyltransferase -1.32 / -1.64 -1.52 / -1.57 - gi|338805200 1625 Ribonuclease T2 5.15 / 3.58 2.93 / 2.83 - gi|338805583 2034 Cytosol aminopeptidase -1.63 / -1.57 - -

! 91! The influence of Heterotrophic Locus N. G5-7 community Accession # Tag: Description winogradskyi (Rep1/Rep2) of G5-7 Nit79A3 (Rep1/Rep2) (Rep1/Rep2) gi|338806285 2776 Lipase class 3 2.18 / 2.13 3.12 / 2.53 - gi|338805484 1928 ATP-dependent Clp protease proteolytic subunit - -1.73 / -1.69 -1.73 / -1.69 gi|338806518 3014 Alpha-glucan phosphorylase - -2.03 / -1.79 -2.03 / -1.79 gi|338806969 3490 Peptidase M4 thermolysin - -1.29 / -1.73 -1.29 / -1.73 Nitrogen Assimilation and Amino Acid Synthesis / Metabolism gi|338804310 673 Aminotransferase class V -1.69 / -2.03 -2.57 / -2.67 - gi|338805895 2365 Glutamine synthetase, type I -1.40 / -1.54 -2.09 / -2.21 - gi|338806043 2515 Nitrogen regulatory protein P-II -2.38 / -2.11 -1.52 / -1.52 - gi|338806861 3379 Alanine dehydrogenase/PNT domain protein -1.35 / -1.51 -2.15 / -2.01 - gi|338803826 161 Glycine hydroxymethyltransferase - -1.60 / -1.69 -1.60 / -1.69 gi|338804474 841 Threonine synthase - -1.65 / -1.48 -1.65 / -1.48 gi|338804826 1224 Glycine dehydrogenase (decarboxylating) subunit 1 - -1.99 / -2.26 -1.99 / -2.26 gi|338804828 1226 Glycine dehydrogenase (decarboxylating) subunit 2 - -1.75 / -1.93 -1.75 / -1.93 gi|338805276 1704 Glutamate-1-semialdehyde 2,1-aminomutase - -1.50 / -1.57 -1.50 / -1.57 gi|338805542 1989 ATP phosphoribosyltransferase - -1.53 / -1.42 -1.53 / -1.42 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N- gi|338806404 2895 - -1.10 / -1.55 -1.10 / -1.55 succinyltransferase gi|338806806 3317 Glu/Leu/Phe/Val dehydrogenase - -1.52 / -1.50 -1.52 / -1.50 Replication, transcription, translation and nucleotide biosynthesis gi|338804081 440 Integration host factor subunit beta -1.83 / -1.25 - - gi|338804209 572 Dihydroorotase, multifunctional complex type -1.53 / -1.23 - - gi|338804216 579 PpiC-type peptidyl-prolyl cis-trans isomerase 1.45 / 1.67 1.72 / 1.94 - gi|338804452 819 50S ribosomal protein L7/L12 -1.93 / -1.79 -2.32 / -1.86 - gi|338804691 1073 Peptidyl-prolyl cis-trans isomerase cyclophilin type 2.34 / 1.56 - - gi|338805468 1912 S-adenosylmethionine synthase -1.43 / -1.66 -2.19 / -2.42 - gi|338803669 2 DNA polymerase III, beta subunit - -1.62 / -1.52 -1.62 / -1.52 gi|338806332 2823 Translation elongation factor Tu - 2.14 / 1.63 2.14 / 1.63 gi|338806665 3172 Single-strand binding protein - -1.67 / -1.87 -1.67 / -1.87

! 92! The influence of Heterotrophic Locus N. G5-7 community Accession # Tag: Description winogradskyi (Rep1/Rep2) of G5-7 Nit79A3 (Rep1/Rep2) (Rep1/Rep2) Miscellaneous gi|338806370 2861 Tetratricopeptide TPR_2 repeat-containing protein 1.56 / 1.45 - - gi|338804646 1025 Peptidoglycan-binding domain-containing protein - 2.79 / 2.73 2.79 / 2.73 Bacteriophage related gi|338804625 1004 Phage tail sheath protein, putative - 2.07 / 1.56 2.07 / 1.56 Hypothetical and unknown function gi|338804162 524 Hypothetical protein 3.26 / 2.88 - - gi|338804471 838 Hypothetical protein 2.57 / 3.81 3.59 / 2.96 - gi|338804597 974 Hypothetical protein -2.70 / -1.67 -1.25 / -1.68 - gi|338804892 1299 Hypothetical protein -1.72 / -1.58 -2.25 / -1.91 - gi|338805151 1573 Protein of unknown function 1.87 / 2.02 - - gi|338805427 1867 Hypothetical protein 1.75 / 2.01 3.14 / 2.13 - gi|338805428 1868 Hypothetical protein 1.64 / 1.70 2.17 / 1.54 - gi|338805889 2359 Hypothetical protein 1.85 / 2.49 - - gi|338805974 2444 Hypothetical protein 2.21 / 1.84 2.10 / 1.59 - gi|338806490 2984 Hypothetical protein 1.57 / 1.44 - - gi|338806603 3107 Hypothetical protein 1.96 / 1.53 - - gi|338806604 3108 Hypothetical protein 2.26 / 1.38 - - gi|338806650 3154 Hypothetical protein -1.52 / -1.65 -2.00 / -2.54 - gi|338804639 1018 Hypothetical protein - 1.72 / 1.61 1.72 / 1.61 gi|338805185 1609 Hypothetical protein - -1.49 / -1.89 -1.49 / -1.89 gi|338805805 2265 Hypothetical protein - 1.79 / 1.41 1.79 / 1.41 gi|338806101 2576 Hypothetical protein - -1.47 / -1.77 -1.47 / -1.77 gi|338806350 2841 Hypothetical protein - -1.69 / -1.79 -1.69 / -1.79 gi|338806966 3487 Hypothetical protein - -2.31 / -2.69 -2.31 / -2.69

! 93! abundance. Many of these proteins have cellular functions related to energy generation, oxidative stress response, nutrient binding, macromolecule degradation or carbon metabolism (Table 16).

The ATP synthase F1 complex proteins (subunits α, β, γ and δ) decreased in abundance (Table

16). In addition the abundance of the F1 subunit ε and the Fo subunit b decreased but the p-values were higher than 0.01 (Table 14). Proteins related to the cellular oxidative stress response changed in a site-specific manner. The cytoplasmic proteins superoxide dismutase, rubrerythrin, thiol peroxidase and two chaperonins decreased in abundance while in the periplasmic space several di-heme cytochrome peroxidases increased in abundance (Table 16). The aminopeptidase A and polyribonucleotide nucleotidyltransferase (cytoplasmic proteins) decreased in abundance and a periplasmic lipase and ribonuclease increased in abundance (Table 16). Nutrient binding proteins for sulfate (periplasmic sulfate binding protein), copper (CopC) and iron (extracellular solute-binding protein family 1) all increased in abundance (Table 16). Proteins related to carbon metabolism such as phosphoglycerate kinase, ribulose-phosphate 3-epiperase and the flavoprotein subunit of succinate dehydrogenase complex showed a lower abundance in the presence of N. winogradskyi (Table 16). In addition, two separate clusters of 2 hypothetical proteins each increased in abundance (1867/1868 and 3107/3108) (Table 16).

The influence of the G5-7 community on the proteome of Nitrosomonas sp. Is79. Seventy (70) Nitrosomonas sp. Is79 proteins changed in abundance in the enrichment culture G5-7 relative to the pure culture of Nitrosomonas sp. Is79 (Table 15 and 16). The abundance of 23 proteins increased and 47 decreased, 55 of which had an annotated function. Most changes in the Nitrosomonas sp. Is79 proteome observed in the presence of N. winogradskyi were also observed in the enrichment culture G5-7: a decrease of the ATP synthase

F1 complex proteins (subunits α, β and γ), an increase in nutrient binding proteins and a decrease of both cellular oxidative stress response proteins and macromolecule degrading enzymes in the cytoplasm but an increase in the periplasm. In addition proteins involved in the ammonia oxidation pathway increased in abundance in the enrichment culture G5-7: ammonia monooxygenase (Amo) subunits A and B (AmoA and AmoB), ORF5 (AmoE), hydroxylamine dehydrogenase (Hao), cytochrome P460 and cytochrome c-554 (Table 15 and 16).

The influence of heterotrophic bacteria on the proteome of Nitrosomonas sp. Is79.

! 94! In the enrichment culture G5-7 Nitrosomonas sp. Is79 was influenced by the heterotrophic community as well as the NOB N. winogradskyi. Therefore the effect of heterotrophic bacteria on the proteome of Nitrosomonas sp. Is79 was determined indirectly by subtracting the proteins whose abundance changed in the presence of N. winogradskyi from the proteins whose abundance changed in the enrichment culture G5-7. One major effect of the presence of the heterotrophic community was the increase of proteins related to the ammonia oxidation pathway. In the presence of the heterotrophic community decreased the abundance of proteins related to carbon metabolism, triosephosphate isomerase, enolase, phosphofructokinase, succinyl-CoA ligase subunit β, transketolase and the ribulose bisphosphate carboxylase oxygenase (RuBisCO) large chain and small chain subunits (Table 16). In addition several proteins involved in the glycine/serine/threonine amino acid synthesis and metabolism pathways such as glycine dehydrogenase subunits 1 and 2, threonine synthase and glycine hydroxymethyltransferase and the first enzyme in histidine biosynthesis, ATP phosphoribosyltransferase, were less abundant (Table 16).

! 95! Discussion Individually as well as together, N. winogradskyi and the heterotrophic community of the enrichment culture G5-7 positively affected the growth of Nitrosomonas sp. Is79 (Fig. 5, 6 and 7; Table 12 and 13). When grown in co-culture with N. winogradskyi or as part of the enrichment

+ culture G5-7, Nitrosomonas sp. Is79 grew at a lower steady state NH4 concentration in continuous culture and at a higher growth rate in batch culture (Fig. 5, 6 and 7; Table 12 and 13). We used this collection of cultures to investigate the mechanisms underlying the positive influence of NOB and heterotrophic bacteria on the growth of Nitrosomonas sp. Is79. iTRAQ proteomics was used to determine the effect of N. winogradskyi and the heterotrophic community of G5-7 on the proteome of Nitrosomonas sp. Is79 when grown in continuous cultures (Table 16).

- In the AOB-NOB co-cultures NO2 was removed by N. winogradskyi resulting in a relief - of the NO2 induced toxic effects and stress on Nitrosomonas sp. Is79 (105, 124, 147). Direct comparison of the proteome of Nitrosomonas sp. Is79 when grown as a pure culture and when co-cultured with N. winogradskyi was used to determine possible mechanisms contributing to the positive influence of N. winogradskyi on Nitrosomonas sp. Is79. Site-specific changes of several proteins related to intracellular oxidative stress were observed in the presence of N. winogradskyi (Table 16). The abundance of proteins responsible for the neutralization of reactive oxygen species (ROS) decreased in the cytoplasm and increased in the periplasm, while intracellular chaperonins also decreased (Table 16). The differential abundances of proteins involved in the

- response to oxidative and nitrosative stress indicate that the removal of NO2 from the growth environment of Nitrosomonas sp. Is79 resulted in a reduction of stress for the AOB. The reduced intracellular stress response very likely freed up energy in the form of PMF, ATP or NADPH, because Nitrosomonas sp. Is79 would have less intracellular damage requiring repair. More energy could then be used for biomass production and growth of Nitrosomonas sp. Is79. The relief of oxidative stress is also the underlying mechanism for the positive interaction between the photoautotroph Prochlorococcus and the marine heterotroph Alteromonas sp. (275). Nitrosomonas sp. Is79, like N. europaea, utilizes proton motive force (PMF) to produce both ATP and NADPH (126, 276). NADPH is generated through reverse electron flow, which makes the production of NADPH more energetically expensive for AOB than the production of ATP (126, 276). The abundance of several components of the F1-ATP synthase were lower

! 96! when Nitrosomonas sp. Is79 was co-cultured with N. winogradskyi (Table 16) indicating that Nitrosomonas sp. Is79 has reduced the production of ATP in the presence of N. winogradskyi. This observation suggests that Nitrosomonas sp. Is79 can use more of the PMF produced by ammonia oxidation for the production of NADPH. The increased generation of reducing power and lower production of ATP would favor biosynthetic pathways such as carbon fixation. This finding is in contrast to a study focusing on the transcriptome of N. europaea in the presence and absence N. winogradskyi (147). Transcripts of ATP synthase subunit genes were up-regulated in N. europaea when grown in co-culture with N. winogradskyi (147). These differences could be due to species-specific responses of AOB to NOB, different tolerances to oxidative and nitrosative stress of N. europaea and Nitrosomonas sp. Is79, differential experimental conditions, or dynamic transcriptional, translational, and post-translational levels of control (270). The abundance of the nitrite reductase NirK increased when Nitrosomonas sp. Is79 was

- co-cultured with N. winogradskyi (Table 16). In contrast, nirK in N. europaea is induced by NO2 accumulation and down-regulated in the presence of N. winogradskyi (124, 147). Nitrosomonas sp. Is79 lacks most of the nitrogen oxide metabolism genetic inventory present in N. europaea including the rest of the nirK operon therefore it is possible that NirK plays different roles in Nitrosomonas sp. Is79 and N. europaea (2,116). During ammonia oxidation NirK is hypothesized to act as an electron sink and prevent the build up of the toxic intermediate hydroxylamine (69, 70). nirK has been shown to be essential for efficient ammonia oxidation in N. europaea with ammonia oxidation rates decreasing in nirK-mutants compared to the wildtype

- (70). Since the NO2 concentration in the chemostats was below 15 µM when N. winogradskyi was present (Fig. 7; Table 13) our results suggest that NirK might be important for more efficient

- ammonia oxidation rather than in the response to NO2 in Nitrosomonas sp. Is79. Future work is required to decipher the exact and possibly multiple roles of NirK in different AOB. In summary, the shifts in the proteome of Nitrosomonas sp. Is79 in the presence of N.

- winogradskyi were very likely caused by the reduction of the NO2 stress and resulted in a decrease of energy spent in stress response, more energy efficient ammonia oxidation and a subsequently higher growth rate. This conclusion is supported by the lower Ks (Table 12) and the faster growth rate of Nitrosomonas sp. Is79 when co-cultured with N. winogradskyi in batch culture (Fig. 5 and 6).

! 97! The heterotrophic community in the enrichment culture G5-7 consisted of Alpha-, Beta- and Gammaproteobacteria and Bacteriodetes (Table 10). Members of these phyla were also found in co-cultures of AOB and cyanobacteria with heterotrophic bacteria (7, 8, 277). The addition of heterotrophic isolates as single isolates and as a community both resulted in enhanced growth rates of Nitrosomonas sp. Is79 (Fig. 5 and 6). Positive effects of heterotrophic bacteria on the growth and metabolism of autotrophic bacteria have been observed in methanotrophs (278) and cyanobacteria (277, 279). The underlying mechanisms of these interactions are broad and often not limited to distinct phylogenetic groups because the heterotrophic bacteria are hypothesized to provide a common cellular metabolite, eliminate toxic compounds or reduce oxidative stress on autotrophs (277-281). The effect of the heterotrophic bacteria on the proteome of Nitrosomonas sp. Is79 was determined indirectly. The effect of N. winogradskyi on the proteome of Nitrosomonas sp. Is79 was subtracted from the effect of the community present in the enrichment culture G5-7, resulting in a list of proteins very likely impacted by the presence of the heterotrophic bacteria in the enrichment culture (Table 16). When Nitrosomonas sp. Is79 was growing as part of the enrichment culture G5-7, several proteins directly related to the ammonia oxidization pathway were more abundant relative to the Nitrosomonas sp. Is79 and N. winogradskyi co-culture (Table 16). Nitrosomonas sp. Is79 relies on ammonia oxidation for its energy generation and a higher abundance of proteins involved in ammonia oxidation could result in the production of more energy and subsequently faster growth. The increased growth rate and ability of Nitrosomonas sp. Is79 in the enrichment culture

+ G5-7 to grow at a lower steady state NH4 concentration (Fig. 5, 6 and 7; Table 13) support this conclusion. In addition, to the enhanced ability to oxidize the primary energy source, 6 proteins related to amino acid synthesis and metabolism decreased in abundance when Nitrosomonas sp. Is79 was grown as part of the enrichment culture G5-7 (Table 16). This suggests that when grown in co-culture with other heterotrophic bacteria, Nitrosomonas sp. Is79 might down-regulate specific amino acid biosynthetic pathways, because they receive exogenous amino acid(s) or amino acid metabolite(s) from the heterotrophic community. In support, certain amino acids have previously been shown to enhance the growth rate of N. europaea (254, 255). The positive interaction between Nitrosomonas sp. Is79 and the diverse group of heterotrophic bacteria (Table 10 and 11)

! 98! could therefore involve the exchange of amino acids and/or amino acid metabolites for small carbon compounds from the autotrophic AOB. Amino acids have been shown to facilitate bacterial interactions between Porphyromonas gingivalis and Treponema denticola (282). P. gingivalis produces and secretes glycine in the presence of, T. denticola, which positively affects the growth of the latter (282). The shifts in the proteome of Nitrosomonas sp. Is79 grown as a part of G5-7 are a combination of the effects of both N. winogradskyi and heterotrophic bacteria (Table 16). The combination of positive interactions resulted in the lowest Ks (Table 12), the lowest steady state

+ NH4 concentration (0.75-1.22 µM) (Table 13) and the fastest growth rate in batch culture (Fig. 5) of Nitrosomonas sp. Is79 in the enrichment culture G5-7. The increased abundance of proteins involved in the ammonia oxidation pathway and the decreased abundance of ATP synthase proteins provides some insight into how interactions between the AOB and both groups had a positive effect on growth of Nitrosomonas sp. Is79. In summary, Nitrosomonas sp. Is79 positively interacted with both N. winogradskyi and heterotrophic bacteria in community cultures resulting in less stress and more energy for the AOB. This type of controlled community culture experiment begins to bridge the gap between pure culture physiologic experiments and natural environmental microbial community studies.

Acknowledgements We thank Rachael Morgan-Kiss and Andor Kiss for technical support with the Next Generation Sequencing and critical reading of the manuscript. The work was supported by start- up funds and Funds from the Committee of Faculty Research at Miami University, by the National Science Foundation grant DEB-1120443 to AB and by a grant from the 5th Framework program of the European Commission (ICON EKW-CT-2000-00054) to NPR.

! 99! SUMMARY Nitrification is a two step microbially mediated process that is responsible for oxidizing

+ - the most reduced form of nitrogen, ammonium (NH4 ), to the most oxidized form, nitrate (NO3 ), - via nitrite (NO2 ). Ammonia-oxidizing bacteria (AOB) and archaea (AOA) perform the first half - of aerobic nitrification, the oxidation of ammonia (NH3) to NO2 (26), while nitrite-oxidizing - - bacteria (NOB) oxidize NO2 to NO3 to complete nitrification (27, 28). All ammonia-oxidizers are chemolithoautrophic and fix carbon for growth through either the Calvin cycle or a modified hydroxypropionate/hydroxybutyrate pathway (102, 106, 134). Ammonia-oxidizers produce relatively low amounts of energy compared to heterotrophic bacteria (127) and about 80% of the energy generated by AOB is used to fix CO2 (135, 283). Since the discovery of AOA in 2004, comparative biochemical and physiological studies with AOB have been ongoing (88, 102, 106). AOA and AOB comparisons have been mainly focused on niche differentiation and investigating their co-existence across many different terrestrial and aquatic environments (108, 109, 184, 217). AOB are the most well studied nitrifiers, having been discovered in the late 19th century by Sergei Winogradsky and since isolated from numerous terrestrial and aquatic environments around the world (2, 140-142). The majority of AOB laboratory-based studies to date have been conducted with pure cultures enriched, isolated and grown at environmentally irrelevant high

+ concentrations of NH4 (112, 125, 284, 285). This cultivation and growth strategy allows for the production of large amounts of biomass, but it leaves a large part of the environmental AOB diversity uncultivated and uncharacterized (169, 209). It also overlooks any interactions between

+ AOB and other community members. Select AOB are adapted to the low NH4 concentrations + found in a variety of natural aquatic and terrestrial environments and are inhibited by high NH4 concentrations (61, 62, 169, 209). Studying AOB with different physiological adaptations, in

+ particular AOB adapted to low NH4 concentrations, is crucial to investigate how ecological disturbances such as eutrophication may affect AOB populations.

+ The goal of this dissertation was to characterize AOB adapted to low NH4 concentrations. + Several AOB adapted to low NH4 concentrations were genomically and physiologically characterized to identify conserved genomic inventory, which correlates with their observed physiological adaptations. In addition, an isobaric tag for relative and absolute quantification (iTRAQ) LC MS/MS proteomics approach was utilized to investigate how the proteome of

! 100! + Nitrosomonas sp. Is79, an AOB adapted to low NH4 concentrations, responds to continuous co- culture with NOB or heterotrophic bacteria.

AOB Physiological and Genomic Comparison Physiological adaptations of Nitrosomonas cluster 6a and 7 AOB to abiotic features that are in constant flux in natural aquatic environments were investigated through a series of growth experiments. The growth rates of Nitrosomonas cluster 6a but not Nitrosomonas cluster 7 AOB

+ - were sensitive to high NH4 and NO2 concentrations. In addition, the ability of select

Nitrosomonas cluster 6a AOB to utilize urea as a source of NH3 was observed. All AOB investigated, achieved their highest growth rates when cultured at a neutral pH with atmospheric levels of O2. + This dissertation produced the first sequenced genome of an AOB adapted to low NH4 concentrations, Nitrosomonas sp. Is79 (2). It also is the first report of the draft genome sequences of: Nitrosomonas oligotropha, Nitrosomonas sp. Is341, Nitrosomonas sp. JL21, Nitrosomonas sp. HPC101 and Nitrosomonas sp. GH22. In silico whole genome comparisons between Nitrosomonas cluster 6a and 7 AOB were conducted to identify genomic traits that were conserved within and between each cluster. We hypothesized that Nitrosomonas cluster 6a and 7 AOB would have separate and identifiable conserved genomic traits that would lend explanations of or further hypotheses about their observed physiological adaptations to low and

+ high NH4 concentrations, respectively. Nitrosomonas cluster 6a AOB genomes lack the extensive nitrogen oxide metabolism

- inventory needed to handle NO2 and nitrosative stress that is present in Nitrosomonas cluster 7 - AOB. The nitrogen oxide metabolism inventory that is absent, includes the NO2 or nitric oxide (NO) responsive transcriptional regulator nsrR and the NO reductase sNOR gene cluster. Select Nitrosomonas cluster 6a AOB also lack the NO reductase norCBQD gene cluster, which suggest these AOB have no enzymatic pathway to reduce NO to nitrous oxide (N2O). Together, this - suggests that Nitrosomonas cluster 6a AOB have different response mechanisms to NO2 and nitrosative stress than Nitrosomonas cluster 7 AOB. Nitrosomonas cluster 7 AOB are adapted to

+ - high NH4 concentrations and may combat high NO2 or NO concentrations on a more frequent basis than Nitrosomonas cluster 6a AOB. To date, the only studies investigating the production of nitrogenous greenhouse gases (GHGs) such as NO or N2O by AOB have utilized the

! 101! Nitrosomonas cluster 7 AOB Nitrosomonas europaea (69, 70). However, the physiologic characteristics of N. europaea are often broadly applied to all Betaproteobacterial AOB. The results presented here suggest that select Nitrosomonas cluster 6a AOB cannot produce N2O or they possess a novel and currently unidentified N2O production pathway.

The ability of AOB to utilize urea as a source of NH3 has been proposed as an + advantageous adaptation for AOB adapted to low NH4 concentrations (80, 119, 209). However, it has been previously observed (80, 140, 209) and is reported here that although no

Nitrosomonas cluster 7 AOB characterized to date can utilize urea as a source of NH3, it is not a conserved adaptation among Nitrosomonas cluster 6a AOB. AOB able to utilize urea encode a urea transporter and the urease gene cluster (ureDABCEFG). The presence or absence of urea amidolyase (UAL) encoding genes did not correlate with urea utilization, even though it facilitates growth on urea as the sole nitrogen source in other microorganisms (225, 226). Overall, this dissertation highlights how the utilization of physiological growth experiments in tandem with in silico genomic comparisons is a powerful characterization tool.

Outlook and future directions With the large amount of anthropogenic nitrogen added to the environment each year (~150 Tg), being able to predict, model and control nitrification in both natural and engineered environments is becoming increasingly important (54). The widespread eutrophication of terrestrial as well as inland and coastal aquatic environments from increased fertilizer application (59, 60) can result in large population shifts in microbial communities (63-65). Culture independent techniques such as meta-proteomics, -transcriptomics and -genomics are often utilized to investigate ecosystem wide changes in microbial communities (186-192). When applied, these –omic techniques identify overarching changes in microbial communities, but are often not sensitive enough to detect species level differences between closely related species (12, 168). Investigating and correlating physiological adaptations within functional groups of microorganisms, such as AOB, to genomic inventory can provide insights into how environmental disturbances may influence microbial community succession and potentially overall ecosystem function. This tandem use of physiological and genomic comparison could also be used to differentiate the adaptations of closely related species from other functional

! 102! groups of autotrophic microorganisms that are often grouped together based solely on their principal metabolic process, such as methanogens or sulfur-oxidizers. Characterizing species-level genomic and physiological differences within functional groups of microorganisms can be used to predict how the overall function of an ecosystem might change based on microbial community succession. For example, Nitrosomonas cluster 7 AOB but not all Nitrosomonas cluster 6a AOB encode a collection of nitrogen oxide metabolism genes responsible for the production of nitrogenous GHGs. As more environments are affected by eutrophication, succession from Nitrosomonas cluster 6a AOB to Nitrosomonas cluster 7 AOB will become more prevalent. This species-level succession will not change the ability of the microbial community to oxidize NH3 but it may lead to an increase in nitrogenous GHG ecosystem emissions. In order to investigate if Nitrosomonas cluster 6a and 7 AOB produce different amounts of NO or N2O, a microrespiratory chamber with fitted injection lids and the appropriate sensors could be used to measure NO and N2O emissions under a variety of different abiotic environmental conditions (70). If used in tandem with transcriptomics and/or proteomics, this type of experimental setup could also identify previously uncharacterized genes involved in

NO or N2O production. Higher NO and N2O emissions from Nitrosomonas cluster 7 AOB could represent an environmental problem moving forward, as the eutrophication of historically oligotrophic environments continues. Microbial GHG emission studies may become more common in the near future as their contribution to overall atmospheric GHG concentrations has been a point of debate in recent years (70, 206, 286).

The Effect of Bacterial Community Members on the Proteome of Nitrosomonas sp. Is79 In addition to characterizing the adaptation of several Nitrosomonas cluster 6a AOB to

+ low NH4 concentrations, ammonia-oxidizing community and co-cultures were utilized to + investigate how NOB and heterotrophic bacteria affect the growth and proteome of the low NH4 adapted AOB, Nitrosomonas sp. Is79. This dissertation contains one of only a handful of proteomic studies ever conducted with AOB and is the first that investigated an AOB other than N. europaea or Nitrosomonas eutropha (72, 260-262). We utilized the following cultures derived from the ammonia-oxidizing enrichment culture G5-7 (164): (1) Nitrosomonas sp. Is79, (2) the NOB Nitrobacter winogradskyi, and (3) several newly isolated, co-cultivated heterotrophic bacteria. When co-cultured with either N. winogradskyi or heterotrophic bacterial community

! 103! members the growth rate of Nitrosomonas sp. Is79 increased. In addition, Nitrosomonas sp. Is79

+ was able to grow more efficiently and achieve a lower steady state NH4 concentration when grown as a continuous culture in the presence of N. winogradskyi. To further investigate the observed positive affects of N. winogradskyi and heterotrophic bacteria on the growth of Nitrosomonas sp. Is79, an isobaric tag for relative and absolute quantification (iTRAQ) proteomics approach was used. The proteome of Nitrosomonas sp. Is79 was determined when grown in 3 different steady state continuous cultures: (1) in pure culture, (2) co-cultured with N. winogradskyi and (3) as part of the community culture G5-7. The effect of N. winogradskyi on the proteome of Nitrosomonas sp. Is79 was determined through direct comparison of the proteome of Nitrosomonas sp. Is79 between the pure Nitrosomonas sp. Is79 culture and the co-culture of Nitrosomonas sp. Is79 with N. winogradskyi. The effect of the G5-7 heterotrophic bacterial community on the proteome of Nitrosomonas sp. Is79 was determined indirectly, by subtracting the proteomic effect of N. winogradskyi observed in the co-culture of Nitrosomonas sp. Is79 with N. winogradskyi from the proteomic effect observed in the enrichment culture G5-7. In both cases, all proteome shifts reported were relative to the proteome of Nitrosomonas sp. Is79 grown as a pure culture. We hypothesized that N. winogradskyi and the heterotopic bacterial community in G5-7 would each cause the proteome of Nitrosomonas sp. Is79 to shift independently and that both proteome shifts would involve unique proteins related to energy generation and the cellular oxidative stress response. When co-cultured with N. winogradskyi, the proteome of Nitrosomonas sp. Is79 responds

- to the reduction of NO2 stress, which results in a decreased amount of energy spent on the cellular stress response and more efficient ammonia oxidation (Fig. 9). The presence of heterotrophic bacterial community members led to increases in the abundance of ammonia oxidation related proteins and the decrease of proteins involved in amino acid synthesis and metabolism (Fig. 9). The shifts in the proteome of Nitrosomonas sp. Is79 observed when grown as a part of the enrichment culture G5-7 are a combination of the individual effects of both N. winogradskyi and the heterotrophic bacterial community (Fig. 9). Together, this illustrates how simultaneously interacting with N. winogradskyi and heterotrophic bacteria had the largest effect on the growth of Nitrosomonas sp. Is79 (Fig. 9). Now that an experimental setup and protocol has been established using iTRAQ proteomics with AOB, more detailed experiments focusing on the interactions between AOB and heterotrophic bacteria can be performed. Investigation of the

! 104!

Figure 9. Model of the effect of heterotrophic bacteria (left) and N. winogradskyi (right) on the proteome of Nitrosomonas sp. Is79. Select proteins that increased (white) or decreased (grey) in abundance are shown. Proteins with dashed lines are for illustrative purposes only and were not differently regulated. AMO, ammonia monooxygenase; HAO, hydroxylamine dehydrogenase; c554, cytochrome c554; cm552, cytochrome cm552; Q/QH2, ubiquinone- ubiquinol pool; bc1, cytochrome bc1 (complex III); c552, cytochrome c552; HCO (c)aa3, cytochrome (c)aa3; ATP Syn, ATP synthase; NADH DH, NADH dehydrogenase; PMF, proton- motive force; P460, Cytochrome P460; GDH1, glycine dehydrogenase subunit 1; GDH2, glycine dehydrogenase subunit2; ThrS, threonine synthase; GlyT, glycine hydroxymethyltransferase; GroEL, 60 kDa chaperonin; GroES 10 kDa chaperonin; TDST, 2,3,4,5-tetrahydropyridine-2,6- dicarboxylate N-succinyltransferase; AADH, Glu/Leu/Phe/Val dehydrogenase; PRT, ATP phosphoribosyltransferase; GluA, Glutamate-1-semialdehyde 2,1-aminomutase; SOD, superoxide dismutase; Rr, rubrerythrin; NirK, nitrite reductase; TPX, thiol peroxidase; Ahp, alkyl hydroperoxide reductase; CcP, di-heme cytochrome c peroxidase; Clp, Clp protease subunit; αGP, α-glucan phosphorylase; PNPT, polyribonucleotide nucleotidyltransferase; CA, cytosol aminopeptidase; CopC, copper resistance protein; ESBP, extracellular solute-binding protein family 1; PSBP, sulfate ABC transporter periplasmic binding protein; HCBR, hemolysin- type calcium-binding region; Lipase, lipase class 3; T2, ribonuclease T2.effect of heterotrophic bacteria on the proteome of Nitrosomonas sp. Is79, when not in the presence of a NOB would be the next step in characterizing AOB-heterotroph interactions.

! 105!

Effect of the G5-7 Community Effect of Effect of Heterotrophic Bacteria N. winogradskyi

HAO 4e- NirK Periplasm c554 P460

NH3 - - NH OH NO2 + PMF c552 NO2 NO + 1/2O2 2 PMF - PMF 2e- 2e PMF PMF cm552 - AMO 4e HCO ATP - - - bc 2e NADH 2e 2e 1 Syn (c)aa3 DH Q/QH2 Q/QH2

2H+ H+ H+ H+ ADP ATP + 1/2O2 H2O NAD NADH Energy Generation + 2H+

SOD GroES TPX Rr CcP GroEL

H O H O H2O2 H2O Oxidative Stress 2 2 2

Clp αGP PNPT CopC ESBP Glycogen Glycogen Protein Amino Acids (n) (n-1) mRNA mRNA PSBP HCBR + + (n) (n-1) - - PO4 Glucose 1-PO 4 Lipase T2 Nutrient Binding & Macromolecule Degradation

Glu/Lys/Phe/Val Catabolism

AADH Glu/Ser/Thr Metabolism GlyT ThrS

Gly/His/Lys Biosynthesis GDH1 PRT GDH2 TDST

Amino Acid Synthesis and Metabolism Cytoplasm

! 106! effect of heterotrophic bacteria on the proteome of Nitrosomonas sp. Is79, when not in the presence of a NOB would be the next step in characterizing AOB-heterotroph interactions.

Outlook and Future Directions As mentioned previously, genomic and physiological comparisons used in tandem provide a great experimental setup for investigating the biochemical potential of individual pure microbial cultures. However, pure culture growth under laboratory conditions is not representative of native environmental growth conditions. To begin to bridge the gap between culture independent studies that give an overview of environmental microbial communities in situ and pure culture studies that focus on single isolated microorganisms; we utilized co-culture, community cultures and enrichment cultures. These types of cultures allow for the investigation of community interactions in cultures that have a manageable number of community members compared to in situ community growth or interaction experiments. As -omic technologies have improved and more sequenced microbial genomes become available, the number of studies investigating microbial communities and their interactions through proteomics (14, 18-20, 192, 199) or transcriptomics (147, 189) has risen. The growth physiology and proteome of Nitrosomonas sp. Is79 when co-cultured with N. winogradskyi and heterotrophic bacteria reported here may provide a more representative view of the physiology and growth of Nitrosomonas sp. Is79 in situ. At the time of writing this dissertation, a study was published investigating the interactions between N. europaea and N. winogradskyi grown in continuous co-culture using transcriptomics (147). The findings support the proteome data and conclusions presented in this dissertation on the response of AOB to co-culture with NOB (147). Together, these studies suggest that the AOB-NOB interaction is to a certain degree conserved across nitrifying species and the results presented here are not species-specific responses that cannot be applied further. However, to investigate the bigger picture of natural microbial community interactions, more in-depth analyses are needed to investigate the interactions between AOB and heterotrophic bacteria. Aside from the continuous culture setup utilized in this report and in Perez et al., 2015, spent media experiments as well as the use of dialysis bags could be utilized to investigate AOB- heterotroph interactions. Dialysis bags can be used to physically separate microbial populations to distinguish interactions that require physical contact such as formation of biofilms or

! 107! multicellular clusters from interactions solely dependent on secreted or diffusible factors (287, 288). Several novel dialysis bag experimental set-ups were constructed and tested for use in batch culture experiments over the course of this dissertation, because it has been previously shown that co-cultures of AOB and NOB cluster together in multicellular flocs (10, 146, 289). However, heterotrophic bacterial contamination was a major problem in these systems due to the long incubation times of AOB and the high susceptibility of AOB cultures to heterotrophic contamination. Investigating how AOB interact with NOB and heterotrophic bacteria in nitrifying communities as mentioned above, is essential to determine how these communities function in situ. It is unknown if AOB, AOA or NOB select for particular heterotrophic bacteria or heterotrophic bacteria with particular functions in nitrifying communities. A metagenomic sequencing approach could be utilized to characterize the heterotrophic bacterial populations within nitrifying enrichment cultures from a variety of environments. The presence of conserved trends based on: species, genus, phylum or metabolic function could be investigated through principal coordinate analysis. In addition, the possible selection of NOB by AOB or AOA could be investigated through competition based growth experiments. Quantitative polymerase chain reaction (qPCR) could be utilized to follow the populations of multiple NOB populations within an AOB or AOA pure or enrichment culture (290). Determining how nitrifying communities form may shed light on possible conserved or general interactions occurring in these communities across natural and engineered ecosystems.

Overall Summary This dissertation presents the genomic characterization of observed physiological

+ adaptations of Nitrosomonas cluster 6a and 7 AOB to low and high NH4 concentrations respectively. The large differences in the genomic inventory for N2O generation between the AOB presented here, illustrate how physiological traits that have previously been broadly applied to large numbers of species based on their shared principal metabolism is not supported. As more whole genome sequences become available and in silico gene annotations improve, a theme of characteristic modularity has started to emerge (291). Characteristic modularity is the concept that the genomic content that drives a particular physiological characteristic (module) can be transferred individually and independently of other modules. This implies that the

! 108! presence of one physiologic characteristic or module in a species does not guarantee the presence or absence of any other module in that species (291). Modular metabolic characteristics disrupt the old way of thinking about many chemolithoautotrophic microorganisms, which have historically been grouped together based on their ability to carry out one specific function, regardless of other metabolic differences they may have. The second focus of this dissertation was to characterize the effect of NOB and heterotrophic bacteria on the proteome of the Nitrosomonas cluster 6a AOB, Nitrosomonas sp. Is79. Overall, the bacterial community members were able to decrease the amount of oxidative and nitrosative stress in Nitrosomonas sp. Is79, which led to more efficient ammonia oxidation and faster growth rates of Nitrosomonas sp. Is79. The ability of bacterial community members to decrease oxidative stress in autotrophic bacteria has been seen previously (275). Although this study, along with Perez et al., 2015, has investigated interactions within nitrifying communities, further characterization of the interactions between nitrifiers and heterotrophic bacteria are needed. In a time where, -omic based projects have become commonplace (13, 14, 16, 167), classical microbial physiologic experiments are needed more than ever to interpret and effectively utilize the often-overwhelming amount of sequence data. This dissertation illustrates that a middle ground between pure culture physiological experiments and large -omic projects is needed in the characterization of ammonia-oxidizers. Co-cultures and enrichment cultures are powerful tools when utilized with either -omic experiments or the characterization of pure cultures as reported here because they allow for the detection species-level differences in community cultures. The collection of genomic, proteomic and physiological observations

+ presented here is currently the most in-depth look at AOB adapted to low NH4 concentrations to date.

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