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Cultivation and genomic sequencing of novel nitrate-oxidizing bacteria

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Cultivation and genomic sequencing of novel nitrate-oxidizing bacteria
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Boddicker, Andrew Michael ( author )
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Nitrification is a critical, rate-limiting step in the removal of nitrogen pollution from freshwater systems. Nitrite-oxidizing bacteria carry out an important regulatory function in the environment by converting nitrite to nitrate, which is utilized by many microbes to facilitate nitrogen loss to the atmosphere. Recent research revealed that nitrite oxidation is carried out by a very diverse group of bacteria, with equally diverse metabolisms. Few representatives have been cultured in the lab due to long incubations and heterotrophic contaminants. Here we describe the cultivation of novel freshwater Nitrotoga species, as well as the first reported Nitrotoga genomes. Four enrichment cultures were initiated from water column and sediment samples from Colorado rivers, each of which enriched a novel Nitrotoga species. Genomic DNA sequencing and assembly revealed highly conserved 16S rRNA gene sequences, but a surprisingly broad diversity among the rest of the genomes. A survey of the core metabolism of each Nitrotoga species revealed genes for growth on organic carbon and growth at low oxygen concentrations, implicating potential to maintain function across a range of environments including biofilms, soil aggregates, and wastewater treatment plants. This work considerably expands our knowledge of Nitrotoga and improves our understanding of their role in the environment. Future efforts will focus on the response of Nitrotoga to environmental change.
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by Andrew Michael Boddicker.

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Full Text
CULTIVATION AND GENOMIC SEQUENCING OF NOVEL NITRITE-OXIDIZING BACTERIA
by
ANDREW MICHAEL BODDICKER B.A., University of Delaware, 2014
A thesis submitted to the
Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science
Biology Program
2017


This thesis for the Master of Science degree by Andrew Michael Boddicker has been approved for the Biology Program by
Annika C. Mosier, Chair Chris Miller Rebecca Ferrell Michael Greene
Date: July 29, 2017


Boddicker, Andrew Michael (M.S., Biology Program)
Cultivation and Genomic Sequencing of Novel Nitrite-Oxidizing Bacteria Thesis directed by Assistant Professor Annika C. Mosier
ABSTRACT
Nitrification is a critical, rate-limiting step in the removal of nitrogen pollution from freshwater systems. Nitrite-oxidizing bacteria carry out an important regulatory function in the environment by converting nitrite to nitrate, which is utilized by many microbes to facilitate nitrogen loss to the atmosphere. Recent research revealed that nitrite oxidation is carried out by a very diverse group of bacteria, with equally diverse metabolisms. Few representatives have been cultured in the lab due to long incubations and heterotrophic contaminants. Here we describe the cultivation of novel freshwater Nitrotoga species, as well as the first reported Nitrotoga genomes. Four enrichment cultures were initiated from water column and sediment samples from Colorado rivers, each of which enriched a novel Nitrotoga species. Genomic DNA sequencing and assembly revealed highly conserved 16S rRNA gene sequences, but a surprisingly broad diversity among the rest of the genomes. A survey of the core metabolism of each Nitrotoga species revealed genes for growth on organic carbon and growth at low oxygen concentrations, implicating potential to maintain function across a range of environments including biofilms, soil aggregates, and wastewater treatment plants. This work considerably expands our knowledge of Nitrotoga and improves our understanding of their role in the environment. Future efforts will focus on the response of Nitrotoga to environmental change.
The form and content of this abstract are approved. I recommend its publication.
Approved: Annika C. Mosier


ACKNOWLDEGEMENTS
I would like to thank my advisor, Dr. Annika Mosier, for her phenomenal support over the course of this project; I really appreciate all that you do. Thank you to my committee members, Dr. Chris Miller, for your extensive help with bioinformatics, and Dr. Rebecca Ferrell, for your great ideas and contributions. I am also grateful to Adrienne Narrowe, for her outstanding support and for teaching me how to be a better researcher.
I have had the pleasure of working with several other students on this project and would like to thank Nick Deevers, Colin Beacom, Mike Kain, and especially Hannah Clark for starting these cultures. I wouldn't have gotten through this program without the support of friends from the Mosier, Miller, and Roane labs, as well as all of the other graduate students. And a final thank you to my friends and family for their love and support.
IV


TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION....................................................................1
II. MATERIALS AND METHODS...........................................................4
Culture Inoculation and Growth................................................4
Growth Curve..................................................................5
DNA Extraction................................................................5
Sequencing....................................................................6
Metagenome and Nitrotoga Genome Assembly......................................6
Nitrotoga Genome Annotation...................................................8
Nitrotoga Comparative Genomics................................................9
Nitrotoga Phylogenetics.......................................................9
III. RESULTS AND DISCUSSION.........................................................10
Enrichment & Nitrite Oxidation...............................................10
Metagenome Assembly and Binning..............................................10
Nitrotoga Genome Assembly....................................................12
Comparative Genomics.........................................................14
Nitrotoga 16S rRNA Gene......................................................16
Nitrotoga Carbon Metabolism..................................................18
C02 Fixation...............................................................18
Organic Carbon Utilization.................................................19
Nitrotoga Nitrogen Metabolism................................................20
Nitrite Oxidation..........................................................20
Assimilatory and Dissimilatory Nitrogen Metabolism.........................27
Nitrotoga Electron Transport for Energy Conservation.........................28
Nitrotoga Motility and Chemotaxis............................................30
Nitrotoga Membrane Transport, Signaling, and Defense.........................31
IV. CONCLUDING REMARKS.............................................................33
REFERENCES.............................................................................35
APPENDIX: Supplemental Materials.......................................................42
v


CHAPTER I
INTRODUCTION
Ever increasing anthropogenic sources of nitrogen, such as fertilizer and wastewater, have led to environmental risks including eutrophication, acidification, and increased greenhouse gas emissions. Nitrite-oxidizing bacteria (NOB) play a critical role in mitigating the harmful effects of nitrogen pollution by linking nitrogen sources to nitrogen removal processes. Specifically, nitrite pools from natural (e.g., ammonia oxidation) or anthropogenic (e.g., fertilizer) sources are converted into nitrate, which is then removed from the system as inert nitrogen gas via denitrification or anammox. Thus, understanding the physiology, metabolism, and environmental limits of NOB is important for controlling and managing elevated nitrogen in contaminated ecosystems.
Despite the environmental relevance of NOB, the group is understudied in part due to their slow growth in the lab and extended enrichment periods, taking as long as 12 years for isolation (Lebedeva et al. 2008). Assiduous cultivation efforts (Watson and Waterbury 1971; Watson et al. 1986; Alawi et al. 2007; Sorokin et al. 2012; Daims et al. 2015; van Kessel et al. 2015), as well as single-cell (Ngugi et al. 2015) and metagenomic (Pinto et al. 2015) sequencing studies are beginning to illuminate the diversity of NOB. NOB have been found in four phyla and seven different genera, three of which were discovered within the last decade: Nitrotoga, Nitrolancea, and Candidatus Nitromaritima (Alawi et al. 2007; Sorokin et al. 2012; Ngugi et al. 2015). Members of the Nitrobacter and Nitrospira genera have been relatively well studied both in the environment and in culture, but our understanding of nitrite oxidation by members of other genera is deficient. NOB are associated with not only a wide range of phylogenetic diversity, but also physiologic versatility, including complete ammonia oxidation (comammox within the Nitrospira), mixotrophic growth with organic
1


carbon, differences in electron transport, and features that enable niche development in specific
habitats (Starkenburg et al. 2008; Liicker et al. 2010; Liicker et al. 2013; Daims et al. 2015; van Kessel et al. 2015; Ngugi et al. 2015; Daims et al. 2016).
Of the currently described NOB, Nitrotoga have recently become an area of focus for nitrification research due to their ubiquity in natural and engineered environments, including wastewater treatment plants where they may play an important functional role (Liicker et al. 2015). Recent molecular surveys have identified Nitrotoga-Wke sequences in a surprisingly wide range of habitats, including: glacial soils (Sattin et al. 2009; Schmidt et al. 2009; Pradhan et al. 2010; Srinivas et al. 2011), an underground cave (Chen et al. 2009), a freshwater seep (Roden et al. 2012), a drinking water filter (White et al. 2012), a subglacial Antarctic lake (Christner et al. 2014), Yellow Sea seawater (Na et al. 2011), salt marsh sediments (Martiny et al. 2011), rivers (Fan et al. 2016), antibiotic-impacted rivers (Li et al. 2011), and various wastewater treatment facilities (Bereschenko et al. 2010; Karkman et al. 2011; Liicker et al. 2015; Saunders et al. 2015; Ziegler et al. 2016; Yang et al. 2016). The relative proportion of Nitrotoga-Wke sequences in several wastewater treatment facilities and a subglacial lake were as high as 2-13% of the total estimated bacterial community, and were occasionally the only observed NOB (Christner et al. 2014; Liicker et al. 2015; Saunders et al. 2015). The distribution and relative abundance of Nitrotoga species across the globe indicate that these organisms likely play a critical role in global nitrogen cycling in a diverse range of environments; however few studies have attempted to characterize their environmental impact.
Candidatus Nitrotoga arctica, enriched from permafrost affected soils in 2007, was the first identified member of the novel genus Nitrotoga (Alawi et al. 2007). Three other members of the same genus have since been enriched from a wastewater treatment plant, aquaculture system, and coastal sand (Alawi et al. 2009; Hupeden et al. 2016; Ishii et al. 2017). These Nitrotoga enrichments
2


have begun to be characterized physiologically, but there are no confirmed isolates and no available
genomes to date. Without genomic data, our understanding of potential Nitrotoga metabolic versatility and niche adaptation in different environments is lacking.
The four previously reported Nitrotoga enrichments were all derived from cold environments, and were enriched at temperatures between 4C and 17C to improve selection. Nitrotoga sp. HW29 retained about 40% of its nitrite-oxidizing capabilities at 10C, but had an optimal temperature for growth of 22C (Hupeden et al. 2016). The upper limits for growth among the four enrichments (as measured in culture) were 25-29C. Nitrotoga sp. HW29 showed growth at pH as low as 5.7, and slightly acidic media improved purification of the enrichment (Hupeden et al. 2016). Co. Nitrotoga arctica has an intermediate nitrite affinity when compared to Nitrobacter and Nitrospira NOB, and is adapted to low nitrite concentrations (0.3 mM) (Nowka et al. 2015).
Here, we describe the draft genome sequences of four novel enrichments of distinct Nitrotoga species. Each organism contains copies of all essential nitrite oxidation genes, as well as complete electron transport chains, and capabilities for inorganic carbon fixation and organic carbon degradation. The possession of high-affinity terminal oxidases, as well as chemotaxic and oxygen binding molecules, indicate that these Nitrotoga species are likely able to survive in low oxygen environments. These findings extend our understanding of freshwater nitrite oxidation, and form the basis for further experimental work aimed at testing genomic predictions in culture and in the environment.
3


CHAPTER II
MATERIALS AND METHODS
Culture Inoculation and Growth
Enrichment cultures were grown in Freshwater Nitrite Oxidizer Medium (FNOM) with a nitrite concentration of 300 pM. FNOM was prepared by mixing 1 g NaCI, 0.4 g MgCI2-6H20, 0.1 g CaCI2-2H20, 0.5 g KCI, 100 pL 10X vitamin solution (Balch et al. 1979), 1 mL 1M NaHC03, and 300 pL of 1M NaN02 per liter. The pH of the media was lowered to 7.0 using 10% HCI, and it was then autoclaved. After autoclaving, 10 mL of separately autoclaved 4 g/L KH2P04 and 1 mL trace metals solution (Biebl and Pfennig 1978) was sterilely added to the media before storing at 4C in the dark.
Sediment samples (n=2) were taken in February of 2015 from two locations of Cherry Creek in downtown Denver, CO (samples MKT and LAW). Sediment samples were taken aseptically using a cut-off sterile syringe, and returned to the lab on ice. The same day, 0.5 cm of sediment (~3 mL) was well mixed with 10 mL of sterile FNOM, and then 1 mL of the sediment mixture was transferred to 100 mL of sterile media for incubation at room temperature in the dark.
Water column samples (n=2) were taken in May of 2015 from two rivers in a more agriculturally dominated region near Greeley, CO, about 100 km North of Denver, CO (samples CP45 from the Cache La Poudre River and SPKER from the South Platte River). River water was kept on ice in the field and stored at 4C upon return to the lab. After five days, 10 mL of water from each site was transferred to sterile FNOM and allowed to incubate at room temperature in the dark.
Growth of enrichment cultures was regularly monitored using a Griess nitrite color reagent (Griess-Romijn van Eck 1966) composed of 0.5 g sulfanilamide, 0.05 g N-(l-naphthyl) ethylenediamine dihydrochloride, 5 mL 85% phosphoric acid, and MilliQ. water to a final volume of
4


50 mL. The solution was 0.2 pm filter sterilized, and stored in the dark at 4C. Nitrite color reagent
was mixed with the culture separately at a 1:1 or 1:10 ratio.
Standard enrichment cultures were transferred to new media approximately every two weeks. Enrichment was enhanced by serial dilution, rapid transfer as soon as nitrite was starting to disappear, and low volume transfers (as low as 0.1% inoculum). Enrichment for Nitrotoga spp. was later enhanced by moving a subset of cultures to grow at 10C and media pH was dropped to 6.0 before autoclaving (Alawi et al. 2007; Alawi et al. 2009; Hiipeden et al. 2016).
Culture stocks were frozen in 10% DMSO as described by Vekeman et. al (2013), but without TSB addition and only one wash step to reduce loss of cells. Additional aliquots were fixed in 4% paraformaldehyde for future fluorescence in situ hybridization experiments.
Growth Curve
Triplicate cultures of each culture line (CP45, LAW, MKT, and SPKER) were inoculated with 100 pL inoculum into 100 mL fresh media to measure nitrite oxidation rates. Samples of each culture were collected at regular intervals for nitrite measurements. Equal volumes of fresh Griess nitrite color reagent and culture were mixed, and the optical density was measured at 540, 545, and 550 nm using a BioTek Synergy HT plate reader (BioTek, Winooski, VT). Mean max OD was used to calculate nitrite concentrations based on comparison with a standard curve of sterile media ranging from 0-300 pM nitrite with the Gen5 analysis software (BioTek, Winooski, VT). All measurements were made in triplicate.
DNA Extraction
Liquid cultures were filtered through a 0.2 pm Supor 200 filter (Pall, New York, NY), then removed with sterile forceps, cut with a sterile scalpel, and aseptically placed into a Lysing Matrix E Bead Beating Tube (MP Biomedicals, Santa Ana, CA) with 800 pL of lysis buffer (0.75 M Sucrose, 20
5


mM EDTA, 400 mM NaCI, 50 mM Tris (pH 8.4)) and 100 pL of 10% SDS. The solution was vortexed
briefly before bead beating in a FastPrep-24 5G reciprocating homogenizer (MP Biomedicals, Santa Ana, CA) at 5 m/s for 30 seconds. The samples were incubated at 99C in a dry bath for 1-3 minutes before adding 50 pL of 20 mg/mL proteinase K. The samples were then incubated for at least 3 hours (up to overnight) in a rotating hybridization oven at 55C. Cold 100% ethanol (500 pL) was added to the sample in the Lysing Matrix E Bead Beating Tube. DNA was then purified using the DNeasy Blood and Tissue Kit (following manufacturer's instructions for purification) (Qiagen, Hilden, Germany). Isolated DNA was quantified with a Qubit fluorometer (Thermo Fisher, Waltham, MA), following the manufacturer's guidelines for the High Sensitivity dsDNA assay.
Sequencing
Extracted DNA was sequenced at the University of Colorado Anschutz Medical Campus Genomics Core on an lllumina HiSeq 2500 using V4 chemistry (lllumina, San Diego, CA) with 2x125 bp paired end reads. DNA was sheared using a Covaris S220 (Covaris, Woburn, MA) and libraries were prepped with an insert size of 400 bp using an Ovation Ultralow System V2 (No. 0344) kit (Nugen, San Carlos, CA).
Metagenome and Nitrotoga Genome Assembly
Sequencing adapters were removed and reads were quality trimmed and filtered using BBDuk (http://jgi.doe.gov/data-and-tools/bbtools/). Reads with adapter overlaps of at least 8 bp (mink=8) with one allowed mismatch (hdist=l) were trimmed, as well as bases under a Phred score threshold of 20 (qtrim=20) and a minimum final read length of 50 bp (minlength=50). Additional bases beyond 125 bp were removed (ftm=5), while both reads in a mate pair were trimmed (tpe), and merged mate pairs were used to trim adapters based on overlap (tbo). Read distributions were
6


manually checked for quality using FASTQC (https://www.bioinformatics.babraham.ac.uk/proiects/
fastqc/) before and after trimming and filtering.
16S rRNA genes were assembled from the trimmed and filtered reads using the EMIRGE platform (Miller et al. 2011). Taxonomy was assigned via a blastn (BLAST+ 2.6.0) (Camacho et al.
2009) search against the SILVA 16S rRNA database (release 123). The assembled 16S rRNA gene sequences later helped to confirm binning and taxonomic assignments of assembled genomes.
Metagenomic reads from each culture were assembled separately using MEGAHIT (Li et al. 2014) with kmer sizes ranging from 31 to 121, in steps of 10. Reads were mapped to contigs using BBMap (http://igi.doe.gov/data-and-tools/bbtools/) and then binned using MetaBAT (--verysensitive -B20) (Kang et al. 2015). Bin completeness and contamination estimates were calculated using CheckM's lineage workflow (Parks et al. 2015).
Binning was manually refined using the Anvi'o metagenomics pipeline version 2.1.0 (Eren et al. 2015), but instead of submitting multiple metagenome samples, each bin was treated as its own 'metagenome' to confirm bin calls made by MetaBAT and combine bins when necessary. Combined bins were reanalyzed with CheckM to determine if completeness estimates increased, while contamination estimates stayed similar. Genes were called within each bin using Prodigal (Hyatt et al. 2010), and blastp was used to find the best hit for each gene from bins of interest against the UniRef90 database (release 2016_11) (Suzek et al. 2014). Individual contigs with suspect taxonomic results were scrutinized and removed upon later reassembly (see below).
Reads were mapped back to the contigs of all individual bins using BBSplit (http://jgi.doe.gov/data-and-tools/bbtools/), and were used for reassembly with SPAdes 3.9.0 (Bankevich et al. 2012). SPAdes was run under the "careful" setting, with MEGAHIT contigs given as "trusted contigs" and kmer sizes from 31 to 121, in steps of 10. Assembly graphs were visualized
7


using Bandage (Wick et al. 2015) to check for suspected connections between contigs, as well as to
identify potential outlier contigs.
Metagenome-assembled contigs that were not incorporated into larger SPAdes-assembled contigs, or were not contiguous with other contigs, were additionally scrutinized. If contigs had dissimilar best UniRef90 hits, inconsistent blastx hits against the NCBI nr database, and were not found to be present within contigs of other Nitrotoga genomes (from this study), they were removed from the bin before further reassembly.
An iterative reassembly process followed as above while adding reads that mapped to suspected contigs that were 'unbinned' by MetaBAT. This included contigs with 16S rRNA gene sequences, which rarely binned properly. To accomplish this, all metagenome-assembled contigs were searched against the SILVA 16S rRNA database (release 128) (Quast et al. 2013), and those with alignment lengths of >300 bp (or 189 bp for LAW due to Nitrotoga being the best hit for a fragment of that size) that did not fall into appropriate bins, were added to the assembly. The internal BLAST function of Bandage allowed visualization of each 16S rRNA gene contig after reassembly, and only contigs that assembled into the contiguous genome were kept. In all cases, only the Nitrotoga 16S rRNA gene contigs assembled with the rest of the genome. A similar process was followed for adding contigs with nxr gene sequences to the assembly; however this occurred after final annotation. After each iterative assembly, CheckM (Parks et al. 2015) was run to establish any changes in completeness or contamination, and read mappings were visualized using Tablet (Milne et al. 2013) to identify potential misassemblies.
Nitrotoga Genome Annotation
Final reassemblies were filtered to remove contigs <2 kb, all of which had very low and uneven coverage estimates. The Nitrotoga genomes were aligned and contigs reordered with
8


progressiveMauve (Darling et al. 2010), using the Nitrotoga sp. from the MKT culture as a reference
due to its long contig length and simple assembly graph. Genomes were submitted to the DOE-JGI Microbial Genome Annotation Pipeline (MGAP) (Huntemann et al. 2015) for final contig trimming of ambiguous and low-complexity sequences, and functional gene annotation. KEGG pathway maps (Kanehisa and Goto 2000) were used to evaluate the core metabolism of Nitrotoga. Predicted signal peptides and transmembrane regions of annotated proteins were evaluated using SignalP (Petersen et al. 2011) and TMHMM (Krogh et al. 2001).
Nitrotoga Comparative Genomics
Average nucleotide identity (ANI) was calculated between genomes using the IMG website's "Compare Genomes" feature. The pangenomic pipeline from Anvi'o (Eren et al. 2015) was used to cluster coding sequences (CDS) from each Nitrotoga genome in order to establish a "core genome" of genes shared by all four genomes. Unique genes found in each genome were identified, as well as those shared among two or three of the genomes.
Nitrotoga Phylogenetics
Gene and protein sequences of interest were iteratively aligned using MUSCLE (Edgar 2004) or MAFFT (Katoh et al. 2002), and then manually edited. Reference sequences were taken from close relatives and other NOB for 16S rRNA sequence alignment (1,496 bp), as well as other members of the Type II DMSO reductase family for amino acid sequence alignment of nitrite oxidoreductase (1,344 amino acids). Phylogenetic trees were generated using FastTree (Price et al.
2010) with default parameters, including 1,000 resamples without branch length reoptimization. All phylogenetic analyses were run using Geneious version 8.1.8.
9


CHAPTER III
RESULTS AND DISCUSSION
Enrichment and Nitrite Oxidation
Four enrichment cultures were initiated from freshwater sediment and water column samples of Colorado rivers in 2015. Two samples (LAW and MKT) were enriched from the sediment of a heavily contaminated urban creek, and two (CP45 and SPKER) were enriched from the water column of rivers dominated by agriculture and livestock land use. The cultures were enriched for 17 (CP45 and SPKER) or 20 (LAW and MKT) months via serial dilution and rapid transfer to new media before DNA was extracted for sequencing.
Nitrite oxidation rates were determined by measuring nitrite consumption over the course of 22 days (rates calculated during exponential growth). The nitrite oxidation rates of each culture were: 81.7 pM N02/day (SPKER), 132.4 pM N02/day (LAW), 138.6 pM N02/day (CP45), and 141.6 pM N02"/day (MKT). The average consumption rate was 123.6 pM N02"/day, which was similar to values recorded for Co. Nitrotoga arctica (Nowka et al. 2015).
Metagenome Assembly and Binning
Evidence from EMIRGE 16S rRNA gene assembly (Miller et al. 2011) suggested there may be overlap in taxonomy among the four samples based on similar blast hits among cultures to similar taxa. However, further analysis using Anvi'o (Eren et al. 2015) suggested there was very little overlap in terms of genomic diversity between samples, including the four Nitrotoga genomes (Table SI and Fig. SI). Additional attempts at a co-assembly using all reads from all cultures, or all Nitrotoga reads, were unsuccessful as each culture contains unique organisms (Fig. SI).
10


Figure 1. Nitrite oxidation by Nitrotoga enrichment cultures over time. Nitrite concentration was quantified colorimetrically. Each enrichment culture was inoculated in triplicate and logarithmic declines in nitrite concentration were presumed to represent the logarithmic growth phase of the nitrite-oxidizing Nitrotoga species. Error bars show the standard deviation of each measurement.
After curation of the assembly, the samples contained 8 (CP45), 14 (MKT), 23 (LAW), and 32 (SPKER) genome bins, and the Nitrotoga genome bins were the most abundant, or one of the most abundant, organisms based on genome coverage. The majority (47/77) of these bins were determined to be near-complete (>90% based on CheckM estimates). The remaining 30 bins had <90% completeness or had high contamination estimates which necessitates further refinement before analysis. The CheckM lineage workflow was used, which assigns a taxonomic lineage to the genome bin to determine a set of marker genes to use in completeness and contamination
estimates; therefore incomplete bins may be assigned root taxonomy (e.g. k______Bacteria with 104
markers) with fewer marker genes than specific lineages (e.g. c__Betaproteobacteria UID3959 with
11


419 markers), inflating or deflating completeness and contamination estimates. Besides Nitrotoga,
each enrichment culture consisted almost exclusively of Proteobacteria, including at least one highly abundant Pseudomonas spp. as well as a common Methylotenera spp., and several members of the Comamonadaceae family. One genome bin from the SPKER metagenome likely belongs to a protozoan in the Neobodonida order that may prey on bacteria.
The Nitrotoga bins were called as "unresolved Betaproteobacteria" by CheckM. Many of the preliminarily called genes (~50%) from these contigs had best hits to genomes within the Gallionellaceae family when compared using blastp against the UniRef90 database (Suzek et al. 2014), which includes the iron-oxidizing Gallionellea and Sideroxydans genera.
Nitrotoga Genome Assembly
Each enrichment culture was found to contain a unique Nitrotoga spp., which was tentatively named based on sampling location: Nitrotoga sp. CP45, Nitrotoga sp. LAW, Nitrotoga sp. MKT, and Nitrotoga sp. SPKER. The enrichment of Nitrotoga from freshwater has not previously been documented, and no Nitrotoga genomes are publically available. Thus, the genomic characteristics described here will vastly improve our understanding of Nitrotoga and how they may behave in natural and engineered environments.
The Nitrotoga genomes ranged in size from 2.707-2.982 Mbp, with 23-59 contigs and GC content between 47.5% and 48.8% (Table 1). Assembly N50 statistics (69.7-214.8 kbp) were associated with the number of total contigs from each genome (i.e. genomes with fewer contigs typically had higher N50 values). The number of coding sequences ranged from 2,574-2,858 with 36-39 tRNAs encoding all twenty amino acids.
CheckM estimates indicated that the four Nitrotoga genomes were near-complete, based on comparisons to a collection of 419 single-copy gene markers that are conserved within the
12


Betaproteobacteria (UID3959). Nitrotoga sp. LAW, MKT, and SPKER genomes were 98.2% complete,
while the CP45 genome was 97.0% complete due to the loss of three markers that were present in contigs <2 kb which were removed before annotation (PF00731.15 AIR carboxylase; PF01259.13 Phosphoribosylaminoimidazolesuccinocarboxamide synthase; and TIGR02392 alternative sigma factor RpoH).
All four genomes were missing the same five marker genes (TIGR01745 aspartate-semialdehyde dehydrogenase; TIGR01574 tRNA-i(6)A37 thiotransferase enzyme MiaB; PF03618.9 Kinase-PPPase; TIGR01161 phosphoribosylaminoimidazole carboxylase, ATPase subunit; and PF13603.1 Leucyl-tRNA synthetase, Domain 2). A manual search of each HMM using HMMER (Eddy
2011) revealed strong (evalue 13


ttContigs GC % N50 (bp) Mean Length (bp) Median Length (bp) Total Length (bp) CDS tRNA rRNA % Complete % Contam- ination
CP45 59 48.8% 69,731 47,733 35,034 2,816,237 2714 36 6 97.0% 0.30%
LAW 33 48.7% 123,222 85,289 58,971 2,814,534 2730 37 6 98.2% 0.30%
MKT 23 48.6% 214,834 117,704 70,433 2,707,194 2574 39 6 98.2% 0.30%
SPKER 32 47.5% 168,712 93,196 48,253 2,982,257 2858 39 6 98.2% 0.24%
Table 1. Assembly statistics overview for Nitrotoga genomes. SPAdes was used for final assemblies and annotations were completed using the DOE-JGI Microbial Genome Annotation Pipeline. The NXR-containing contig was manually annotated and added to the assembly statistics for each genome. The minimum contig sizes were 2,505 bp (CP45), 4,389 bp (LAW), 4,076 bp (MKT), and 2,458 bp (SPKER).
The Nitrotoga genomes were estimated to contain 0.24-0.30% contamination. Specifically, three of the genomes (CP45, LAW, and MKT) had duplications of the same two markers (PF09976.4 Tetratricopeptide repeat-like domain; PF08340.6 domain of unknown function 1732), while Nitrotoga sp. SPKER only had a duplicate of PF08340.6. One of the copies of each duplicated marker gene had a drastically reduced protein length and weak HMM hit, so they may represent false-positives for gene detection. None of the published Gallionellaceae genomes showed duplications of these genes.
Nitrotoga Comparative Genomics
Average nucleotide identity (ANI) was calculated pairwise between the four Nitrotoga genomes and two most closely related genomes of iron oxidizers from within the Gallionellaceae family: Sideroxydans lithotrophicus ES-1 and Gallionella capsiferriformans ES-2 (Table 2). ANI values between the Nitrotoga genomes and the iron oxidizers ranged from 71.3% to 72.6% identity, indicating a vast phylogenetic distance even from the closest related sequenced genomes. Therefore the iron oxidizer genomes could not be used as a close reference in Nitrotoga genome assembly, and supports the classification of Nitrotoga as a novel candidate genus (Alawi et al. 2007). The Nitrotoga sp. CP45, LAW, and MKT genomes were all closely related to each other, with ANI values ranging from 93.7% to 94.4%. The Nitrotoga sp. SPKER genome, however, was much more distinct,
14


with ANI values ranging from 87.1% to 87.4% when compared to the other three Nitrotoga
genomes. These values are indicative of species level differences between each culture (Richter and Rossello-Mora 2009). The variance in genome content when compared to the conservation of 16S rRNA gene sequences (see below) is also noteworthy, as studies that cluster 16S rRNA sequences alone may under estimate species diversity of the Nitrotoga.
The Anvi'o pangenome pipeline was run on the four Nitrotoga genomes to identify conserved coding sequences (Eren et al. 2015) (Fig. S2). Proteins were clustered using an MCL algorithm to identify homologs at the amino acid level between genomes. Overall, 1,794 protein clusters were identified as a "core genome", meaning all four genomes possessed these coding sequences. Each genome also encoded unique coding sequences not found in any other Nitrotoga genome (CP45: 365 protein clusters, LAW: 348 protein clusters, MKT: 293 protein clusters, SPKER: 626 protein clusters), and 568 protein clusters were found to be shared between two or three genomes only.
15


Nitrotoga sp. CP45 Nitrotoga sp. LAW Nitrotoga sp. MKT Nitrotoga sp. SPKER Sideroxydans lithotrophicus ES-1 Gallionella capsiferrifor-mans ES-2
Nitrotoga sp. CP45 X 93.69% 94.24% 87.43% 72.54% 71.34%
Nitrotoga sp. LAW 93.69% X 94.38% 87.38% 72.36% 71.48%
Nitrotoga sp. MKT 94.23% 94.39% X 87.06% 72.39% 71.47%
Nitrotoga sp. SPKER 87.43% 87.38% 87.06% X 71.94% 71.64%
Sideroxydans lithotrophicus ES-1 72.57% 72.45% 72.46% 72.02% X 72.98%
Gallionella capsiferrifor mans ES-2 71.44% 71.58% 71.71% 71.79% 73.06% X
Table 2. Pairwise average nucleotide identity (ANI) comparisons between enriched Nitrotoga genomes and the two closest available genomes calculated using the IMG website's Compare Genomes feature. These values represent species-level differences between Nitrotoga genomes.
Nitrotoga 16S rRNA Gene
The Nitrotoga genomes contained surprisingly conserved 16S rRNA gene sequences when compared to the genome sequence divergence. The Nitrotoga sp. CP45 and MKT 16S rRNA gene sequences were 99.87% identical with only two mismatches across the entire 1544bp gene, while the most distantly related were MKT and SPKER at 99.42% identity and nine mismatches. The 16S rRNA sequences were also closely related (>88% identity, average 96% identity) to publically available Nitrotoga sequences from the SILVA database (release 128) (Quast et al. 2013).
The top five hits from each genome's 16S rRNA gene sequence were found exclusively from environmental gene surveys (from soil, sediment, river water, and wastewater samples) except for one SPKER hit to the original Candidatus Nitrotoga arctica clone (Table S2). All four 16S rRNA
16


sequences grouped phylogenetically with other Nitrotoga sequences within the Betaproteobacteria
and were divergent from nitrite oxidizers in other phyla (Fig. 2).
*
Bradyrhizobium japonicum USDA 124 [ARFJO1000006)
Nitrobacter hamburgensis X14 (CP000319)
Nitrobacter winograd$kyi Nb-255 (CP00011S)
----- Gallionella capsiferriformans ES-2 [CP002159]
------- Sideroxydans lilhotrophicus ES-1 [CP001965]
Alphaproteobacteria
r Nitrotoga sp. enrichment culture clone HAM-1 (FJ263061)
I Ca. Nitrotoga arnica [DQ839562]
Candidate Nitrotoga sp. done HW29 [KT778545)
If Nitrotoga sp. SPKER
Candidatus Nitrotoga sp. clone AMI [LC190436]
Nitrotoga sp. LAW Nitrotoga sp. CP45 Nitrotoga sp. MKT
Nitrosospira multiformis ATCC 25196 [CP000103]
Nitrosomonas europaea ATCC 19718 [AL954747)
Betaproteobacteria
Nitrosomonas eutropha C91 (CP0004SO)
- Nitrosococcus oceani AFC27 [ABSC01000004]
Nitrococcus mobilis (HM038001)
--------- Thiocapsa sp. KS1 (EF581005)
________________________r------------- Nitrospina gracilis 3/211 (CAQ)01000065]
Gammaproteobacteria
Nitrospinae
- Ca. Nitromaritima sp. A02 [JZKI01000029]
---- Desulfovibrio desulfuricans ND132 [CP003220]
-------------------------- Nitrolancea hollandica Lb (CACS01000547)
4
HI'
Leptospirillum ferriphilum ML-04 [CP002919]
Nitrospira defluvii [FP929003]
Nitrospira moscoviensis (CP011801] |\J j-£ pQg p j pg g
Ca. Nitrospira nitrosa [CZQA01000015]
Figure 2. 16S rRNA sequence phylogenetic tree of NOB and close relatives aligned across 1,496 bp. Members of all seven nitrite oxidizer genera are present, and our 16S rRNA sequences are most similar to other Nitrotoga sequences from enrichment cultures within the Betaproteobacteria.
Other nitrite oxidizers are found in the Alphaproteobacteria (Nitrobacter), Gammaproteobacteria (Nitrococcus and Thiocapsa), Nitrospinae (Nitrospina and Candidatus Nitromaritima), Chloroflexi (Nitrolancea), and Nitrospirae (Nitrospira). Nodes with FastTree support values >50% are shown with a blue circle.
The 5S, 23S, and 16S rRNA genes all assembled together along with two tRNA genes in each genome. However, these genes all had approximately double the coverage of the surrounding contig when reads were mapped back, likely representing a repeat. The operon was split between multiple contigs in the LAW and SPKER genomes resulting in inflated numbers of predicted rRNA genes by MGAP (Table 1), while the same sequence was found in the middle of large contigs for CP45 and
MKT.
17


Nitrotoga Carbon Metabolism
CO? Fixation
All described NOB possess one of two inorganic carbon fixation pathways: the reverse tricarboxylic acid cycle (rTCA) has been found either biochemically or via genome surveys in the Nitrospina, Nitrospira, and Candidatus Nitromaritima (Liicker et al. 2010; Liicker et al. 2013; Ngugi et al. 2015); and the Calvin-Benson cycle dominates in the Nitrobacter, Nitrococcus, and Nitrolancea (Starkenburg et al. 2008; Sorokin et al. 2012). Nitrotoga enrichment cultures were grown in media containing no organic carbon suggesting they also must have a method of fixing inorganic carbon.
Indeed, all four of the Nitrotoga genomes were found to contain genes coding for 12 of 13 enzymes used in the Calvin-Benson cycle to fix carbon dioxide. The missing gene in all four genomes was sedoheptulose-bisphosphatase, which catalyzes the hydrolysis of sedoheptulose 1,7-bisphosphate to sedoheptulose 7-phosphate. Genes coding for this enzyme were also missing from the genomes of another NOB, Nitrobacter winogradskyi, as well as the ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosospira multiformis (Chain et al. 2003; Starkenburg et al. 2006; Norton et al. 2008). Fructose 1,6-bisphosphatase, which typically plays a role in gluconeogenesis and was present in all four Nitrotoga genomes, may fill this role in these genomes, as well as in other organisms (Yoo and Bowien 1995; Wei et al. 2004).
One gene encoding sedoheptulokinase, a bidirectional phosphorylating accessory enzyme (sedoheptulose <-> sedoheptulose 7-phosphate) to the Calvin-Benson cycle, was missing from all four Nitrotoga genomes. Additionally, the SPKER genome was missing genes encoding for xylose-5-phosphate/fructose-6-phosphate phosphoketolase, which play a role in the Calvin-Benson cycle, but are not critical to carbon fixation. The fructose-6-phosphate phosphoketolase catalyzes the same reaction as transketolase, which was present in all four Nitrotoga genomes, and the xylose-5-
18


phophate regenerates glyceraldhyde-3P from xylulose-5P, which can also be accomplished via other
enzymes of the Calvin-Benson cycle.
All four Nitrotoga genomes contained two copies of the ribulose(-5-)-phosphate 3-epimerase enzyme, and SPKER contained two copies of transketolase genes, both of which play roles in the Calvin-Benson cycle and the pentose phosphate pathway, but other key genes were missing from the pentose phosphate pathway. The CP45, LAW, and MKT genomes all had two copies of both large and small ribulose 1,5-bisphosphate carboxylase subunits (RuBisCO), the key enzyme for carbon dioxide fixation, while the SPKER genome had a single copy.
Organic Carbon Utilization
In addition to carbon fixation, all four Nitrotoga genomes had genes encoding proteins for complete glycolysis and pentose phosphate pathways, but not gluconeogenesis. A phosphoenolpyruvate carboxylase gene was missing from all four genomes, which catalyzes the first step of gluconeogenesis (oxaloacetate phosphoenolpyruvate). All other enzymes in gluconeogenesis are the same as those used in glycolysis, except for fructose 1,6-bisphosphatase, which was present in all four genomes but may play a role in carbon fixation rather than gluconeogenesis (see above). Additionally, genes for glucose transport enzymes were missing from all four Nitrotoga genomes.
All four Nitrotoga genomes had genes encoding enzymes that generate acetyl-CoA from pyruvate, as well as an acetyl-CoA synthetase, which produces acetyl-CoA directly from acetate. All Nitrotoga genomes contained the full suite of genes for the TCA cycle, but were missing both key enzymes of the reverse TCA cycle for C02 fixation, ATP-citrate lyase and 2-oxoglutarate:ferredoxin oxidoreductase, as used by Nitrospira defluvii (Liicker et al. 2010). These complete pathways
19


suggest that Nitrotoga species are likely capable of heterotrophic growth on organic carbon, which
has also been reported in a wide variety of other NOB (Daims et al. 2016).
Nitrotoga Nitrogen Metabolism
Nitrite Oxidation
All described NOB genomes (from the Nitrobacter, Nitrospina, Nitrococcus, Nitrospira, Nitrolancea, and Candidatus Nitromaritima genera) contain genes necessary to form a functional nitrite oxidoreductase (NXR), a three-subunit enzyme (nxrA-alpha subunit, nxrB-beta subunit, nxrC-gamma subunit) that oxidizes nitrite to nitrate, stripping two electrons for use in energy conservation. NXR belongs to the Type II DMSO reductase family of molybdenum enzymes (Liicker et al. 2010; Daims et al. 2016), in which the active site of the alpha subunit contains a molybdenum-bismolydopterin-guanine-dinucleotide (Mo-bisMGD) motif, as well as one iron-sulfur cluster (Magalon et al. 2011; Hille et al. 2014; Daims et al. 2016). The beta subunit contains four iron-sulfur clusters for electron conduction away from the alpha subunit towards the membrane-integral gamma subunit, which is less well understood and is thought to contain up to two heme groups that may be involved in electron transfer to a cytochrome c for delivery to the electron transport chain (Liicker et al. 2010; Magalon et al. 2011; Daims et al. 2016).
The most prominent difference in the NXRs of NOB is the orientation of the enzyme in relation to the cell membrane. Members of the Nitrobacter, Nitrococcus, and Nitrolancea have been shown to possess cytoplasmic-facing NXR (Spieck et al. 1996; Starkenburg et al. 2006; Sorokin et al. 2012), while members of the Nitrospira and Nitrospina have been shown to have periplasmic-facing NXR (Spieck et al. 1998; Liicker et al. 2010; Liicker et al. 2013; Koch et al. 2015). Candidatus Nitromaritima was presumed to have a periplasmic NXR due to predicted signal peptides on the alpha subunit for excretion via the twin-arginine translocation (Tat) pathway (Martinez-espinosa et
20


al. 2007; Sargent 2007; Ngugi et al. 2015). Nitrotoga were also predicted to have a periplasmic-
facing NXR (Nowka et al. 2015). Oxidation kinetics associated with NXR orientation have been established (Nowka et al. 2015) and have implications in ecological niche formation, as bacteria with cytoplasmic-facing NXR typically dominate in relatively high-nitrite environments over their periplasmic-facing counterparts.
In three of the Nitrotoga genomes (CP45, LAW, and MKT), a single 8.7 kb contig had strong hits to available Type II DMSO HMMs for the alpha (e-value le-78), beta (e-value le-77), and gamma subunits (e-value le-39) (TIGR03477-TIGR03479), as well as a designated chaperone, nxrD (e-value le-29) (TIGR03482), that is similar to TorD used in molybdenum cofactor assembly and protein folding (Magalon et al. 2011; Liicker et al. 2013; Hille et al. 2014; Ngugi et al. 2015). Together, these form an nxrDCBA operon. Each contig also encoded a putative 70 amino acid protein of unknown function, and a 341 amino acid protein with conserved domains related to iron-transfer P-loop NTPases, which are required for cytosolic Fe-S cluster assembly (factor NBP35), both located upstream of the nxr genes. CP45 also had two additional hypothetical proteins, one on each end of the contig.
Reads mapped from each end of the NXR contig (in the LAW and MKT genomes) to two other contigs within the assembly, likely representing a repeated region with two distinct locations within each genome. The CP45 NXR contig, however, did not have mapped reads leading to other contigs, and the ends of the contig had much lower read coverage than the interior, indicating that more sequence surrounding this region may be missing. The CP45, LAW, and MKT NXR-containing contigs had similar GC content and approximately double coverage estimates when compared to the rest of their respective genomes, supporting the presence of gene duplications with very high
21


similarity. In all three cases, mapped reads did not indicate any single nucleotide polymorphisms
(SNPs) present in the contig, likely representing two identical copies.
The SPKER genome did not have a single contig corresponding to all NXR genes, but rather the genes were scattered across several contigs. The SPKER NXR contigs assembled with the rest of the genome, with connections to four other contigs, but coverage estimates indicated this genome may have three copies of the nxr genes. The four genes (nxrDCBA) assembled in the same order as all other genomes, but the contig was lacking copies of the two other genes (FeS cluster assembly factor NBP35 and 70 amino acid hypothetical protein) found in the same contig from all three other Nitrotoga genomes. A copy of the FeS cluster assembling factor NBP35 gene was found on a connected contig, but only at single coverage. Mapped reads to this contig also indicated that there may be two different nxrA sequences due to the presence of SNPs. Previous reassemblies using contigs below the coverage threshold for this genome assembled two different copies of nxrA, one with single coverage and one with double coverage. Flowever, the final reassembly resulted in only one nxrA sequence as there was not enough divergence in read mapping to isolate different copies. The nxrDCB genes always assembled at approximately triple coverage when compared to the rest of the genome.
The putative NXR alpha subunits from CP45, LAW, and MKT had a Tat signal peptide on the N-terminal as predicted by the SignalP 4.1 online server (Petersen et al. 2011), which supports the excretion of this enzyme to the periplasm (Sargent 2007) The SPKER NXR alpha subunit was lacking a Tat signal peptide, although an alignment of the four copies showed that the first 14 amino acids may be missing, as the entire peptide was otherwise aligned without gaps (Fig. 3). The putative beta subunit was lacking a signal peptide, but may be excreted via a 'hitch-hiker' method as described previously in other NOB (Martinez-espinosa et al. 2007; Liicker et al. 2010; Pester et al. 2013).
22


The putative gamma subunit of each genome had an N-terminal signal peptide, which was
previously observed in the genomes of Nitrospina gracilis and Candidatus Nitromaritima spp. (Liicker et al. 2013; Ngugi et al. 2015). However, in both cases the predicted signal peptide cleavage site was located in the middle of a predicted transmembrane region, and previous studies had confirmed the membrane-association of the NXR complex in other NOB genera, indicating that these gamma subunits were not fully translocated into the periplasm (Liicker et al. 2010). In the case of Nitrotoga, the putative nxrC gene was located directly next to the alpha and beta subunit genes, but did not contain any predicted transmembrane regions (by InterProScan). This subunit is predicted to belong to the heme-binding members of the Type II DMSO reductase family, however it is more similar to the soluble gamma subunit of ethylbenzene dehydrogenase than the membrane-bound subunit of NXR (Kloer et al. 2006; Magalon et al. 2011). Much less is known about the structure and function of the NxrC subunits, as gene copies even within the same genome can vary in size, identity, and structure. Nitrospira defluvii has four putative nxrC genes scattered across the genome with b or c-type cytochromes which are 30-60 kDa in size when translated (Liicker et al. 2010), while Nitrospina gracilis has four different putative NxrC subunits as well as four versions of an "alternative NxrC" with 1-3 cytochrome c binding sites and a heme-fo domain (Liicker et al. 2013).
Classical NXR enzymes that have been described likely pass electrons stripped from nitrite to a cytochrome c carrier for transport to the terminal oxidase. The potential for a soluble NXR complex may have different implications in electron transport, and more biochemical investigation is warranted. Each Nitrotoga genome encodes multiple cytochrome c genes that may be involved in the shuttling of electrons from NXR or the alternative complex III to the terminal oxidase.
23


NxrA [Nitrotoga sp. CP45]
NxrA [Nitrotoga sp. LAW]
NxrA [Nitrotoga sp. MKT]
NxrA [Nitrotoga sp. SPKER]
NxrA [Nitrospira defluvii]
NxrA [Nitrospira defluvii]
NxrA [Ca. Nitromaritima A02] NxrA [Ca. Nitromaritima C22] NxrA [Nitrospina gracilis]
NxrA [Nitrospina gracilis]
NxrA [Nitrobacter winogradskyi] NxrA [Nitrobacter winogradskyi] NxrA [Nitrococcus mobilis]
NxrA [Nitrococus mobilis]
NxrA [Nitrolancea hollandica]
NxrA [Nitrotoga sp. CP45]
NxrA [Nitrotoga sp. LAW]
NxrA [Nitrotoga sp. MKT]
NxrA [Nitrotoga sp. SPKER]
NxrA [Nitrospira defluvii]
NxrA [Nitrospira defluvii]
NxrA [Ca. Nitromaritima A02] NxrA [Ca. Nitromaritima C22] NxrA [Nitrospina gracilis]
NxrA [Nitrospina gracilis]
NxrA [Nitrobacter winogradskyi] NxrA [Nitrobacter winogradskyi] NxrA [Nitrococcus mobilis]
NxrA [Nitrococus mobilis]
NxrA [Nitrolancea hollandica]
285
285
285
285
285
285
285
285
285
285
285
285
285
285
285
MMEIKNNIGRRS FLKL SA MMEI KNNI gHs FLKL SA MME I KNNI gBs FLKL:SA
--------------KLSA
---MQVSVSfflQFLKI SA
----mmqlsMqflkvsa
-mrl:
-mrl:
-mrl:
srSq:
3RRQ
NRRK
NRRK
nrrk:
FLQVSA
|KFLQVSA
FLQVSA
70 FLCAPNDTHNCLLKAHVKNDW 115 WEPRICNKGM
70 FLCAPNDTHNCLLKAHVKNDW 115 WEPRICNKGM
70 FLCAPNDTHNCLLKAHVKNDW 115 WEPRICNKGM
70 FLCAPNDTHNCLLKAHVKNDW 115 WEPRICNKGM
70 WCCS PNDTHGCRVRAFVRNGW 115 HNPRMCLKGF
70 WVCS PNDTHACRIRAFVRNGW 115 HNPRMCLKGF
70 YCCSPNDTHQCRVRGFVRNGIL 115 WNPRMCLRGM
70 YCCSPNDTHQCRVRGFVRNGIL 115 WNPRMCLRGM
70 YCCS PNDTHQCRVRGFVRNGIL 115 WNPRMCLRGM
70 YCCS PNDTHQCRVRGFVRNGIL 115 WNPRMCLRGM
70 sthgvncTggcswaiyvkdgii 115 YE PRGCQRGI
70 sthgvncJggcswaiyvkdgii 115 YE PRGCQRGI
70 STHGVNCTGGCSWAIYVKDGII 115 YEPRGCQRGI
70 70 sthgvncTggcswaiyvkdgii 115 115 YEPRGCQRGI

DSYSWHT
DSYSWHT
DSYSWHT
DSYSWHT
SNYTWHG
NNYTWHG
SNYTWHG
SNYTWHG
SNYTWHG
SNYTWHG
LAPGCPMVSGHD LDYE 430: LAPGCPMVSGHi LDYE 430: LAPGCPMVSGhI LDYE 430: LAPGCPMVTGH1 LDYE 430: QDPSHPFWNGtI CDVD 430: QDPSQPWWNGTl CDVD 430: QAPGHSWTHGMMSDID 430: QAPGHSWTHGMmSDID 430: QAPGHSWTHGMMSDID 430: QAPGHSWTHGMQTSDID 430:
YKLADLTHHLKVMKPGEHPTMPPAFQA YKLADLTHHLKVMKPGEHPTMPPAFQA YKLADLTHHLKVMKPGEHPTMPPAFQS YKLAELTHHLKVMKPGEGPTMPPAFQS
YKFPDF----------------SKSYS
YQVPDF----------------TKSYS
YKAQQL---------------PKDGFT
YKAQQL---------------PKDGFT
YKNQPL---------------PKDGFT
YKNQPL---------------PKDGFT
SF|DWYA|LPTSFPEIWGDpTDVCE SfIdWYaIlPTSFPEIWGdHdVCE 430: NR VARY SFIDWYaIlPNSFPEIWGdBdVCE 430: EEVKKY
430: NR VARY--------------KDVEN
------------KDVEN
------------KKQEN
SFHDWYAIlPNSFPEIWGDQTDVCE 430: EEVKKY-
-KKQEN
SF(DWYABLPNS FPFVWGDHBDVCF 430: NRINRY-----------------KDVEN
Figure 3. Protein alignments of NxrA catalytic subunit. Sequence number is based on the alignment with NxrA of other nitrite oxidizers, a gap is left between predicted periplasmic-facing subunits (top) and cytoplasmic-facing subunits (bottom). Bright green highlighting represents a twin-arginine translocation signal peptide. Yellow highlighting represents Fe-coordinating residues for an Fe-S cluster. Blue residues represent conserved residues for nitrite/nitrate binding and active site formation, and dark green residues represent a molybdenum coordinating aspartate residue (Martinez-espinosa et al. 2007; Liicker et al. 2010). A conserved threonine residue is replaced with an asparagine in Nitrospira spp. and a methionine in our Nitrotoga genomes. An insertion of 14 amino acids in all Nitrotoga genomes is shown in gray highlighting.
Amino acid alignments of the putative Nitrotoga NXR alpha and beta subunits against other
known NXR proteins revealed conservation of residues that are critical for substrate binding and Fe-S cluster organization in nitrite oxidoreductases and nitrate reductases (Martinez-espinosa et al. 2007; Liicker et al. 2010) (Fig. 3, Fig. 4). However, the alpha subunits were only approximately 35%
identical to reference protein sequences, while the beta subunits were between 30-45% identical to reference protein sequences. When compared to each other, the reference sequences were approximately 64% identical on average.
24


NxrB [Nitrotoga sp. CP45] 30 NxrB [Nitrotoga sp. LAW] 30 NxrB [Nitrotoga sp. MKT] 30 NxrB [Nitrotoga sp. SPKER] 30 NxrB [Nitrospira defluvii] 30 NxrB [Ca. Nitromaritima A02] 30 NxrB [Ca. Nitromaritima C22] 30
NxrB [Nitrococcus mobilis] 30 NxrB [Nitrobacter winogradskyi] 30 NxrB [Nitrobacter winogradskyi] 30 NxrB [Nitrolancea hollandica] 30
NxrB [Nitrotoga sp. CP45] 214 NxrB [Nitrotoga sp. LAW] 214 NxrB [Nitrotoga sp. MKT] 214 NxrB [Nitrotoga sp. SPKER] 214 NxrB [Nitrospira defluvii] 214 NxrB [Ca. Nitromaritima A02] 214 NxrB [Ca. Nitromaritima C22] 214
NxrB [Nitrococcus mobilis] 214 NxrB [Nitrobacter winogradskyi] 214 NxrB [Nitrobacter winogradskyi] 214 NxrB [Nitrolancea hollandica] 214
NxrB [Nitrotoga sp. CP45] 460: NxrB [Nitrotoga sp. LAW] 460: NxrB [Nitrotoga sp. MKT] 460: NxrB [Nitrotoga sp. SPKER] 460: NxrB [Nitrospira defluvii] 460: NxrB [Ca. Nitromaritima A02] 460: NxrB [Ca. Nitromaritima C22] 460:
NxrB [Nitrococcus mobilis] 460 NxrB [Nitrobacter winogradskyi] 460 NxrB [Nitrobacter winogradskyi] 460 NxrB [Nitrolancea hollandica] 460
FDLNKCIACQSCTMACKTTWT
FDLNKCIACQSCTMACKTTWT
FDLNKCIACQSCTMACKTTWT
FDLNKCIACQSCTMACKTTWT
FNINRCLACQTCSMADKSTWL
FNTNRCIACQTCTMAHKSTWT
FNTNRCIACQTCTMAHKSTWT
FHLDKCIGCHTCSIACKNIWT 85 FHLDKCIGCHTCSIACKNIWT 85 FHLDKCIGCHTCSIACKNIWT 85 FHLDKCIGCHTCSIACKNIWT 85
EQAQP^^SWS----GNK-----YNG--TTI FED^HLGMNQRI KGYLPDEMDYAHPNLGEDE CLKB ILDGE
EQAQP--~WS---GNK------ YXG-------TTIFEdHlGMNQRIKGYLPDEMDYAHPNLGEDECLkBILDGE
EQAQP-WS-----GNK------YNG---TT I FEdHlGMNQRI KGYLPDEMDYAHPNLGEDE CLkBILDGE
EQAQP]---WSGNK-------1 YNG------TTIFEdHlgmnQrikgylpdemdyahpnlgedeclkS-_ildge
EQVNPGGQVWNVRVGRKHHAPYGVFEG---MTIFDAGAKVGQAAIGYIPTDQEWRFVNIYEDTATSMRALVENID
EQSNPGQNVWNVR-KTSNKAIHGVYEG---VTIFEAPAKIGLNQQAVGYVPTDEEWRFPNFGEDTAHGREFTQSR
EQSNPGQNVWNVR-KTSNKAIHGVYEG---VTIFEAPAKIGLNQQAVGYVPTDEEWRFPNFGEDT AHGREFTQSR
DQEKYKGG- WERDSRGRLKLKLQGRAGALTNIFYNPSLPTLDDYYEPWTYDYKNLFDAPEGDDQ PTARAVSLVT GD DQTKYRGG-WWD-GQRQKSLRLRLQGKWGTLTNIFYNPYLPTLDDYFEPWTYDYQNLITAPLADEQPTARAISMVT GK DQTKYRGG-WWD-GQRQKSLRLRLQGKWGTLTNIFYNPYLPTLDDYFEPWTYDYQNLITAPLADEQPTARAISMVTGK DQSKYRGG-WERT-GDK--IRLKLQGRRAGLANIFFNPYLPTVDDYYEPWTYQYEDLFNAPEGDDQPTARPISLITGK
FFFPRICNHCT FPGCLAACPRKAIYKRQEDGIVLIDASRCRGYRECVAAC PYKKS FYNDTTRTGEKCISCYPKVEAGLM-----TQCVTQCIGKIR
FFFPRICNHCTFPGCLAACPRKAIYKRQEDGIVLIDASRCRGYRECVAACPYKKSFYNDTTRTGEKCISCYPKVEAGLM--------TQCVTQCIGKIR
FFFPRICNHCT FPGCLAACPRKAIYKRQEDGIVLIDASRCRGYRECVAAC PYKKS FYNDTTRTGEKCISCYPKIEAGLM-----TQCTTQCIGKIR
FFFPRICNHCTFPGCLAACPRKAIYKRQEDGIVLIDASRCRGYRECVAACPYKKSFYNDTTRTGEKCISCYPKVEAGLM--------TQCVTQCIGKIR
FYLQRICNHCTYPGCLAACPRKAIYKRPEDGIVLIDQNRCRGYKKCVEQCPFKKPMYRGTTRVSEKCIACYPRIEGKDPLTGGEPMETRCMAACVGKIR
FYLQRICNHCTYPGCLAACPRKAIYKRQEDGIVLIDQSRCRGYKKCVEQCPYKKPMFRGTTRISEKCIACYPRIEGLDPLTEGDQMETRCMAACVGKIR
FYLQRICNHCTYPGCLAACPRKAIYKRQEDGIVLIDQSRCRGYKKCVEQCPYKKPMFRGTTRISEKCIACYPRIEGLDPLTEGDQMETRCMAACVGKIR
FYLPRICNHCLNPGCVAACPTGAIYKRGEDGIVLISQNRCRAWRMCVSGCPYKKTYFNWSTGKSEKCILCYPRLESGQP--------PACFHSCVGRIR
FYLPRICNHCLNPGCVAACPQGALYKRGEDGWLVSQERCRAWRMCVSGCPYKKTYFNWSTGKAEKCILCYPRLESGQP---------PACFHSCVGRIR
FYLPRICNHCLNPGCVAACPQGALYKRGEDGWLVSQERCRAWRMCVSGCPYKKTYFNWSTGKAEKCILCYPRLESGQP---------PACFHSCVGRIR
FYLPRICNHCLNPGCVAACPAGAIYKRGEDGIVLISQEKCRAWRMCVSGCPYKKTYFNWATGKSEKCILCYPRLESGHA--------PACFHSCVGRIR
RICDTFKl
ricdtfk!
ricdtfk!
RICDTFK-
ILEGNET 520 IlEGNET 520 ILEGNET 520 IlEGNET 520
QIVFRYDVIPGPKVFETQIHGKRFDMYNDTV 520 RIIFKWKREPGPKIFETNIHGKKFEMYNDTV 520 RIIFKWKREPGPKIFETNIHGKKFEMYNDTV 520
KIKHVYHKLVAVRMYMRSEQVGDQKEEDV 520 IIRDVYKKLVAVRVYMRSRKVKDIPDEEV 520 IIRDVYKKLVAVRVYMRSRKVKDIPDEEV 520 IVADVYRKLVAVRIFMRSQRVNDIPEAEV 520
YIRQVKNLKTG YIRQVKNLKTG YIRQVKNLKTG YIRQVKNLKTG YIRPAERVNWL*-YVRPEEHPGAI*-YVRPEEHPGAI*-
-AFLTNNS* -AFLTNNS* -AFLTNNS* -AFLTNNS*-
FWPPMERERAMDGELPPLDPYSR-----KAAGGFGFRDAPARRF*
FWP PME RETAVDAL FP QLDP V SHNY PIRKGE VGVG FHTDPARGP FWPPMERETAVDALFPQLDPVSHNYPIRRGAVGVGFHTDPARGP* FVIPAFDREKSIEQTVDPYTH------KPHAGFGFRQAPERRW*
Figure 4. Protein alignments of the NxrB subunits. Sequence numbering is based on the alignment with NxrB of other nitrite oxidizers, a gap is left between predicted periplasmic-facing subunits (top) and cytoplasmic-facing subunits (bottom). Yellow highlighting represents Fe-coordinating residues for four Fe-S clusters. Gray highlighting represents presumptive deletions from all Nitrotoga genomes that are not present in other nitrite oxidizers.
Comparisons of NXR subunits between Nitrotoga genomes indicate a highly conserved
protein structure, and a more divergent nucleotide gene sequence. The alpha subunits of each
genome were at least 98.1% identical across the 1170 amino acid protein, but were as low as 84.1%
identical across the ~3500 bp nucleotide alignment (SPKER vs LAW). Similar patterns were seen with
the beta subunit (>97.9% protein identity, >87.0% nucleotide identity), while the gamma subunit
was more divergent between samples (>92.8% protein identity, >85.3% nucleotide identity). The delta chaperone subunit was highly conserved between the CP45, LAW, and MKT genomes (>99.2% protein identity), but slightly more divergent in the SPKER genome (>96.4% protein identity).
Previously published PCR primers for nxrB genes (nxrB169f/nxrB638r) (Pester et al. 2013) were tested in silico against the Nitrotoga sequences, but both forward and reverse sequences each
25


had five mismatches. The affiliation of the NXR enzyme alpha subunit to putative archaeal NarG
sequences (Fig. 5), along with the mismatched primer sequences and lack of reference sequences, likely explains the lack of Nitrotoga nxr genes observed in environmental molecular surveys, which may suggest that the distribution, diversity, and activity of Nitrotoga on a global scale may be vastly underestimated.
Interestingly, many of the contigs with mapped reads to the NXR contig from each Nitrotoga genome contained genes most closely related to transposases and integrases, meaning the nxr genes could have been horizontally transferred from another organism. Horizontal origins of the NXR in Nitrotoga would support previous reports of the origins of periplasmic-facing NXR. The nxr genes of Nitrospira and Nitrospina were hypothesized to originate from an anaerobic or microaerophilic ancestor that were then passed among the two genera, as well as anammox organisms such as Candidatus Scalindula and Candidatus Kuenenia (Liicker et al. 2010; Liicker et al. 2013). However, this hypothesis was partially based on the presence of a complete rTCA cycle and lack of reactive oxygen defense mechanisms in the Nitrospira and Nitrospina (Liicker et al. 2010; Liicker et al. 2013), neither of which was true for the Nitrotoga genomes (see below).
The cytoplasmic-facing NXRs were hypothesized to be evolutionarily related to anoxygenic phototrophs of the purple sulfur bacteria (Teske et al. 1994), but the recent genomic sequencing of Thiocapsa sp. strain KS1 supports the separate evolution of the Nitrobacter, Nitrococcus, and Nitrolancea NXRs (Hemp et al. 2016). The deeply-branching phylogenetic position of Nitrotoga NxrA protein sequences (Fig. 5) may support another transfer of nxr genes to microaerophilic Gallionellaceae ancestors leading to a separate evolutionary trajectory, or may offer clues to the origins of Nitrospira and Nitrospina NXR, as the Nitrotoga sequences are more closely related to the nearest clade of putative archaeal nitrate reductases (Fig. 5).
26


i Ca. Nitromaritima sp. C22 NxrA [WP_048492943]
* Ca. Nitromaritima sp. A02 NxrA [KMP11423]
Co. Kuenenia stuttgartiensis NarG [CAJ72445] r Nitrospira defluvii NxrA [WP_013249749]
* Nitrospira moscoviensis NxrA [WP_053381277] r~ Halopiger xanaduensis [WP_013880708] Haloterrigena turkmenica [WP_012942830] Hydrogenobacter thermophilus (WP_012964344] Hydrogenobaculum sp. Y04AAS1 [WP_012513384] Acetothermia bacterium NxrA/NarG [BAL57377] Candidate division NC10 bacterium (KRT68883]
Ca. Rokubacteria bacterium [KRT71940] Elusimicrobia bacterium [OGR84873]
Omnitrophica WOR_2 bacterium [OGX52889] i Nitrotoga sp. CP45 NxrA f Nitrotoga sp. SPKER NxrA Nitrotoga sp. LAW NxrA Nitrotoga sp. MKT NxrA Omnitrophica bacterium [OGX04451] Deltaproteobacteria bacterium [OGP09841] Deltaproteobacteria bacterium [OGQ05848] Deltaproteobacteria bacterium [OGQ21411]
Haloarcula hispanica [WP_014030951]
Rhodovulum sulfidophilum DdhA [Q8GPG4] r Ideonella dechloratans ClrA [P60068]
Thauera selenatis SerA [Q9S1H0]
------------- Desulfococcus oleovorans [WP_012173623]
---------------- Azoarcus sp. EB1 EdbA (AAK76387]
Haloarcula marismortui NarG (WP_011223493]
Haloferax mediterranei NarG [CAF21906] r Dechloromonas agitata PcrA [AA049008]
Dechloromonas aromatica PcrA [Q47CW6]
Thiocapsa sp. KS1 NxrA [CRI68048]
Thermus thermophilus HB8 NarG [CAA71210]
Co. Methylomirabilis oxyfera NarG [CBE67843] Nitrolancea hollandica NxrA [JQ279817] Nitrococcus mobilis NxrA [WP_004998773] Nitrobacter winogradskyi NxrA [WP_011315305] Nitrobacter hamburgensis X14 NxrA [ABE64178]
Figure 5. Phylogenetic tree of the alpha subunit of nitrite oxidoreductase enzyme. Nitrotoga NxrA amino acid sequences grouped closest to putative archaeal nitrate reductase (NarG-like) enzymes. References were selected to include members of the Type II DMSO reductase family including both periplasmic and cytoplasmic NxrA. Sequences covering 1,344 amino acids were iteratively aligned with MUSCLE, MAFFT and manual edits before using the default settings for FastTree in Geneious. Nodes with >50% FastTree support values are labeled with a blue circle. Abbreviations: nitrite oxidoreductase (NxrA), nitrate reductase (NarG), dimethyl sulfide dehydrogenase (DdhA), chlorate reductase (ClrA), selenite reductase (SerA), ethylbenzene dehydrogenase (EdbA), perchlorate reductase (PcrA).
Assimilatorv and Dissimilatorv Nitrogen Metabolism
The Nitrotoga genomes encode genes for transport of a variety of nitrogen compounds, including nitrate/nitrite (narK), formate/nitrite, and ammonium (amt). These proteins are likely used to import nitrogenous compounds into the cytosol, where they are used to build amino acids and other biochemical intermediates. Interestingly, a Nitrotoga enrichment culture was found to grow dramatically better when ammonium was added to the culture medium (Ishii et al. 2017), indicating
27


a physiological advantage in the presence of ammonium. Similarly, Nitrolancea hollandica required
ammonium in growth medium to survive, and the genome contained no assimilatory nitrite reductase. All Nitrotoga genomes were found to have a NirBD assimilatory nitrite reductase for formation of ammonia.
A NirK dissimilatory nitrite reductase (N02" NO) was also found in all Nitrotoga genomes. nirK genes have been found in all other NOB genomes except Nitrolancea hollandica (Sorokin et al. 2012; Liicker et al. 2013), but their ultimate role is still unclear. Three of the Nitrotoga genomes (CP45, LAW, and MKT) also encode a nitric oxide dioxygenase, which catalyzes the conversion of nitric oxide to nitrate, and evolutionarily was used as an 02-binding protein similar to hemoglobin (Gardner et al. 1998).
Nitrotoga Electron Transport for Energy Conservation
All four Nitrotoga genomes contained genes for a complete electron transport chain (Fig. 6). NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) were present according to KEGG (Kanehisa and Goto 2000) gene calls via IMG's annotation pipeline. Canonical cytochrome foc2 genes (complex III) were missing from all genomes; however, a suite of genes corresponding to an alternative complex III (ActABlB2CDEF) (Refojo et al. 2012) was found in all genomes. A similar construct was documented in the related iron-oxidizing bacteria of the Gallionellaceae family (Emerson et al. 2013).
A cbb3-type cytochrome c oxidase (complex IV) was found in all four Nitrotoga genomes. This oxidase is a member of the C-class heme-copper oxidases, and has a high affinity for oxygen (Morris and Schmidt 2013). Organisms possessing cbb3-type oxidases are likely capable of growth in microoxic environments (Han et al. 2011), and genes encoding this type of oxidase have been reported in the genomes of the NOB Nitrospina gracilis and the phototrophic nitrite-oxidizer
28


Thiocapsa KS1, enriched from ocean surface waters and activated sludge, respectively (Schott et al.
2010; Liicker et al. 2013; Hemp et al. 2016). Several studies have detected the presence of Nitrospina-Wke and Nitrococcus-Wke bacteria, as well as active nitrite oxidation in marine oxygen minimum zones, although Nitrotoga were not detected (Labrenz et al. 2007; Fuchsman et al. 2011; Fussel et al. 2012). The possession of only a cbb3-type terminal oxidase indicates that Nitrotoga species likely continue nitrite-oxidation and heterotrophic growth at nanomolar 02 concentrations, allowing a potentially wide diversity in habitat. In addition to a cbb3-type terminal oxidase, the Nitrotoga genomes contain several 02 binding proteins including a protoglobin, hemerythrin, and the nitric oxide dioxygenase (see above). 02-binding proteins have been identified in some NOB genomes, but their role is not fully understood. In support of the aerobic nature of Nitrotoga, all four genomes contained necessary catalase and superoxide dismutase genes to combat reactive oxygen species (ROS), as well as a rubrerythrin protein used to combat ROS specifically in anaerobic bacteria.
Interestingly, Nitrotoga sp. SPKER contained an additional fad-type terminal cytochrome oxidase, which was also observed in both Sideroxydans lithotrophicus ES-1 and Gallionella capsiferriformans ES-2 genomes (Emerson et al. 2013). This terminal oxidase could play an additional role in energy conservation from organic carbon sources, as fad-type terminal oxidases only receive electrons from ubiquinones or menaquinones, while cfafa3-type terminal oxidases can receive electrons from cytochromes or members of the quinone pool (Borisov et al. 2011; Morris and Schmidt 2013). Remarkably, a novel "cytochrome fad-like oxidase" was identified in the genome of Nitrospira defluvii that has a putative copper-binding site that may act as a heme-copper oxidase to accept electrons from NXR via a cytochrome c shuttle (Liicker et al. 2010), but a blast search did not identify any homologs in the Nitrotoga genomes. Finally, an F-type ATPase (complex V) is present within all four Nitrotoga genomes for ATP generation (Fig. 6).
29


Figure 6. Tentatively suggested nitrite oxidation and energy conservation model based on genomic data. All Nitrotoga genomes encode NXR subunits (alpha, beta, and gamma), although the gamma subunit is lacking predicted transmembrane regions, indicating it may be soluble. Electron transport chain complexes l-V were present, however a canonical fac^ complex was replaced by an Alternative Complex III. A full oxidative tricarboxylic acid (TCA) cycle is encoded in each genome, as well as glycolytic pathways, suggesting growth on organic carbon is possible. A complex IV fad-type terminal oxidase was present in the Nitrotoga sp. SPKER genome (not pictured), in addition to the cfafa3-type present in all genomes. Dotted lines represent potential electron transfer pathways.
Reverse electron flow to reduce NAD+ to NADH is necessary for inorganic carbon fixation. The alternative complex III, which was found in all Nitrotoga genomes (Fig. 6), is thought to be capable of reverse electron flow, oxidizing cytochrome c and reducing ubiquinone to ubiquinol (Refojo et al. 2012) which is then reoxidized and passed to complex I for NADH production.
Nitrotoga Motility and Chemotaxis
All four Nitrotoga genomes contained all genes necessary for flagella assembly and operation, as well as a signal transduction pathway to stimulate twitching motility via a type IV pilus assembly. Additionally, all genomes had chemotaxis genes, but the SPKER genome was notably missing an aerotaxis receptor (aer) that detects oxygen.
30


Figure 7. KEGG Map of bacterial flagellar assembly. All genes necessary for chemotaxis and production of flagella were present in all Nitrotoga genomes.
Nitrotoga Membrane Transport, Signaling, and Defense
ATP-binding cassette (ABC) transporters were identified for sulfate (cysPUWA), phosphate (pstSCAB), molybdate (modABC), Iron (III) (afuABC), lipopolysaccharide (rfbAB and IptFGB), lipoprotein (lolCED), heme (ccmDCBA), branched-chain amino acids (livKHMGF), phospholipid (mlaCDEBF), and iron complexes (fhuDBC). Interestingly, the genome of Nitrotoga sp. SPKER was missing modB and mode genes, which are critical for obtaining molybdenum for use as a cofactor in NXR.
Complete, or mostly complete two-component signal transduction pathways were present for Cu (l)/Ag (I) ion efflux pumps (cusSRBA), K+ transport pumps (kdpABCDE), as well as nitrogen availability (ntrBC) linked to glutamine synthetase (glnDBLGA). An additional regulator of redox
31


signals (regAB) was present and may signal the expression of both subunits of the cbb3-type terminal
oxidase (ccoNO) in all genomes, as well as the two subunits of the fad-type terminal oxidase (cydAB) in the Nitrotoga sp. SPKER genome.
All Nitrotoga genomes encoded penicillin-binding proteins 1A (mrcA), 2 (mrdA), and 3 (cell division protein Ftsl) which are inhibited by (3-lactams. However, the cells may also exhibit (3-lactam resistance mechanisms via an ampG beta-lactamase induction signal transducer and a nagZ beta-N-acetylhexosaminidase, which helps to recycle cell wall muropeptides (Cheng et al. 2000). Resistance-nodulation-cell division superfamily (RND) efflux pumps were present in all four genomes as multidrug efflux systems. The possession of an antibiotic efflux could increase the competitive advantage of Nitrotoga, particularly in urban or livestock-dominated areas and in wastewater treatment facilities. However, further experimentation is needed to confirm antibiotic resistance, and genome sequences of additional Nitrotoga species are necessary to determine whether or not this is a common trait among all Nitrotoga.
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CHAPTER IV
CONCLUDING REMARKS
Nitrotoga have recently come under the spotlight for nitrification research, and likely play a much larger role in freshwater and engineered environments than originally presumed by nxr PCR studies that likely do not amplify Nitrotoga nxr genes (Pester et al. 2013; Liicker et al. 2015). Here we have described the enrichment and near-complete draft genomes of four novel Nitrotoga species of NOB. These novel species are capable of growth under chemolithoautotrophic conditions, and likely also possess capabilities for small organic molecule oxidation for energy conservation, as detailed in many other nitrite oxidizers (Daims et al. 2016). A mixotrophic metabolism with the use of small organic carbon molecules could help extend the range of sustainable environments, along with a suite of multidrug and metal efflux pumps, which may explain how these organisms were enriched from contaminated urban areas and Nitrotoga sequences have been previously observed in an antibiotic-affected river (Li et al. 2011).
The possession of a cbb3-type terminal oxidase likely allows Nitrotoga to grow at nanomolar 02 concentrations, meaning they may play a vital nitrification role in oxygen limited microenvironments, including biofilms, floes, and aggregates as seen with Nitrotoga in wastewater treatment plants (Liicker et al. 2015). The additional use of putative oxygen binding proteins, and aerotaxic motility, may help coordinate growth in microoxic environments.
Finally, the distribution of Nitrotoga-Wke sequences and Nitrotoga enrichment cultures on a global scale implicates this genus as a crucial player in the Earth's nitrogen cycle. Nitrotoga have been cultivated and/or detected on five different continents, and their ability to dominate nitrite-oxidation at cold temperatures indicates they are likely significant contributors to global nitrite oxidation rates. Our ability to cultivate four novel species of Nitrotoga with surprisingly similar 16S
33


rRNA sequences but dissimilar genomic features also speaks to the astounding diversity that likely
exists in the environment. Future work will include increasingly accurate molecular surveys, competition experiments, and transcriptomics and proteomics to determine Nitrotoga's response to changing environments.
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Appendix
Supplemental Materials
Figure SI. Coassembly of all enrichment cultures with contig splits (~20 kbp fragments split by Anvi'o with respect to gene calls) clustered by differential coverage and tetranucleotide frequency using Anvi'o metagenome pipeline. Regions of overlap between each layer represent contig splits that are likely shared between enrichment cultures. Very few organisms are shared, and the Nitrotoga genomes are highlighted with little overlap between enrichment cultures. The Length ring represents the length of split contigs, while the Parent layer represents splits that originated from the same contig. Each contig is also assigned Taxonomy by Centrifuge (Kim et al. 2016) that is represented by color.
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1
Figure S2. Anvi'o pangenome pipeline was used to identify homologous protein clusters within and between Nitrotoga genomes. Black regions represent protein clusters that are present in the genome. The core genome consists of 1,794 protein clusters found in all four Nitrotoga genomes. Each genome contains unique protein clusters (CP45: 365 protein clusters, LAW: 348 protein clusters, MKT: 293 protein clusters, SPKER: 626 protein clusters). A total of 568 protein clusters were shared among two or three of the Nitrotoga genomes.
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Enrich. Culture Best BLASTN Hit Prior NormPrior
CP45 >KJ161326.1.1708_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;Pseudomonas putida 0.37622 0.33618
CP45 >CP001672.691251.692776_Bacteria;Proteobacteria;Betaproteobacteria;Me thylophilales;Methylophilaceae;Methylotenera;Methylotenera mobilis JLW8 0.1605 0.163306
CP45 >JQ435722.1.1421_Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales ;Phyllobacteriaceae;Aminobacter;Aminobacter aminovorans 0.157609 0.169877
CP45 >FN994922.1.1363_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Pseudomonadaceae;Pseudomonas;uncultured Pseudomonas sp. 0.089176 0.099345
CP45 >G U061253.1.1455_Bacteria; Proteobacteria; Betaproteobacteria; Nitrosomo nadales;Gallionellaceae;Candidatus Nitrotogajuncultured betaproteobacterium 0.083656 0.087152
CP45 >AY212698.1.1518_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Hydrogenophaga;uncultured bacterium 0.081768 0.084607
CP45 >FR733686.1.1474_Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales ;Xanthobacteraceae;Aquabacter;Aquabacter spiritensis 0.023498 0.02721
CP45 >GQ062189.1.1304_Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiale s;Hyphomicrobiaceae;uncultured bacterium 0.022389 0.026204
CP45 >DQ066988.1.1378_Bacteria;Bacteroidetes;Sphingobacteriia;Sphingobacteri ales;PHOS-HE51;uncultured bacterium 0.005184 0.006119
LAW >DQ178219.1.1510_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Pseudomonadaceae;Pseudomonas;Pseudomonas mendocina 0.479532 0.47478
LAW >JX223577.1.1501_Bacteria;Proteobacteria;Betaproteobacteria;Methylophil ales;Methylophilaceae;Methylotenera;uncultured bacterium 0.090594 0.087909
LAW >JX222872.1.1501_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured bacterium 0.078244 0.076178
LAW >GQ339174.1.1504_Bacteria;Proteobacteria;Betaproteobacteria;Nitrosom onadales;Gallionellaceae;Candidatus Nitrotogajuncultured bacterium 0.072743 0.078454
LAW >DQ530075.1.1478_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi alesjComamonadaceaejVariovoraxjVariovorax sp. Cl 17 0.057059 0.056455
LAW >GQ199732.1.1385_Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiale sjBrucellaceaejMycoplanajMycoplana sp. 210_30 0.034259 0.037195
LAW >HQ755752.1.1443_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi alesjComamonadaceaejPseudorhodoferaxjuncultured organism 0.033666 0.034095
LAW >JX221753.1.1496_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadalesjPseudomonadaceaejPseudomonasjuncultured bacterium 0.027371 0.026212
LAW >KC541299.1.1533_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadalesjPseudomonadaceaejPseudomonasjuncultured bacterium 0.023714 0.022844
LAW >KF975518.1.1487_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi alesjBurkholderiaceaejLimnobacterjuncultured bacterium 0.023692 0.024195
LAW >JN644603.1.1538_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia lesjComamonadaceaejDelftiajDelftia lacustris 0.020247 0.019517
LAW >DQ264409.1.1505_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi alesjComamonadaceaejChlorochromatiumjuncultured bacterium 0.019219 0.020033
LAW >AB425068.1.1530_Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenop hilalesjHydrogenophilaceaejThiobacillusjuncultured Thiobacillus sp. 0.017614 0.017764
LAW >JX566634.1.1523_Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; NeisseriaceaejVogesellajVogesella sp. 5137 0.010272 0.010001
LAW >AY328708.1.1482_Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospiril lalesjRhodospirillales Incertae SedisjReyranellajuncultured bacterium 0.007204 0.007372
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LAW >AB682227.1.1456_Bacteria;Bacteroidetes;Sphingobacteriia;Sphingobacteria les;Chitinophagaceae;Lacibacter;Lacibacter cauensis 0.003071 0.003156
LAW >KF851140.1.1502_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured Pseudomonas sp. 0.0015 0.003839
MKT >AJ306778.1.1529_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured bacterium 0.314681 0.29816
MKT >DQ530075.1.1478_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Variovorax;Variovorax sp. Cl 17 0.209116 0.214887
MKT >CP001672.691251.692776_Bacteria;Proteobacteria;Betaproteobacteria;Me thylophilales;Methylophilaceae;Methylotenera;Methylotenera mobilis JLW8 0.109821 0.104125
MKT >EU234184.1.1523_Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomo nadales;Gallionellaceae;Candidatus Nitrotogajuncultured bacterium 0.102424 0.10331
MKT >HM217120.1.1404_Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiale s;Brucellaceae;Ochrobactrum;Ochrobactrum pseudogrignonense 0.053495 0.06948
MKT >FJ230937.1.1465_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Alcaligenaceae;Achromobacter;uncultured bacterium 0.051263 0.049799
MKT >JN869049.1.1525_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Burkholderiaceae;Limnobacter;uncultured bacterium 0.049951 0.04714
MKT >FM201089.1.1328_Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobac terales;Rhodobacteraceae;Gemmobacter;uncultured bacterium 0.041257 0.04435
MKT >DQ264489.1.1514_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Moraxellaceae;Acinetobacter;uncultured bacterium 0.027229 0.026042
MKT >JN868908.1.1476_Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomo nadales;Sphingomonadaceae;Novosphingobium;uncultured bacterium 0.016408 0.015983
MKT >JX644238.1.1455_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Comamonadaceae;Variovorax;uncultured bacterium 0.012092 0.012328
MKT >FIQ640603.1.1397_Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclal es;Rhodocyclaceae;Zoogloea;uncultured bacterium 0.008026 0.010358
MKT >JX222666.1.1497_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Comamonadaceae;Acidovorax;uncultured bacterium 0.003906 0.003711
MKT >JX221874.1.1485_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Comamonadaceae;Variovorax;uncultured bacterium 0.000179 0.000171
MKT >KF441679.1.1525_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;uncultured;Albidiferax sp. 7B-403 0.000152 0.000157
SPKER >EF100238.1.1717_Eukaryota;Excavata;Discoba;Discicristata;Euglenozoa;Kin etoplastea;Metakinetoplastina;Neobodonida;uncultured eukaryote 0.188209 0.189486
SPKER >FN994922.1.1363_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Pseudomonadaceae;Pseudomonas;uncultured Pseudomonas sp. 0.131888 0.132978
SPKER >G U061253.1.1455_Bacteria; Proteobacteria; Betaproteobacteria; Nitrosomo nadales;Gallionellaceae;Candidatus Nitrotogajuncultured beta proteobacterium 0.119056 0.120749
SPKER >AFBH01000032.516.2028_Bacteria;Proteobacteria;Betaproteobacteria;Burk holderialesjComamonadaceaejAcidovoraxjAcidovorax radicis N35v 0.082242 0.074701
SPKER >HM186771.1.1367_Bacteria;Proteobacteria;Betaproteobacteria;Burkholder ialesjBurkholderiaceaejLimnobacterjuncultured bacterium 0.061712 0.062085
SPKER >AB237665.1.1417_Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobact erales;Rhodobacteraceae;Rhodobacter;uncultured bacterium 0.04812 0.0469
SPKER >FJ658806.1.1343_Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;u ncultured bacterium 0.043014 0.044147
SPKER >EF080874.1.1396_Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadalesjPseudomonadaceaejPseudomonasjPseudomonas sp. Lm-3 0.037201 0.037564
SPKER >AAXX01000001.678774.680286_Bacteria;Bacteroidetes;Flavobacteriia;Flav obacterialesjFlavobacteriaceaejFlavobacteriumjFlavobacteria bacterium BAL38 0.034708 0.033661
SPKER >FR744473.1.1424_Bacteria;Proteobacteria;Alphaproteobacteria;Rickettsiale sjRickettsiales Incertae SedisjCandidatus Captivusjuncultured bacterium 0.026418 0.025495
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SPKER >JN868849.1.1525_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Oxalobacteraceae;Paucimonas;uncultured bacterium 0.02632 0.024114
SPKER >AXZP01000076.341.1851_Bacteria;Bacteroidetes;Sphingobacteriia;Sphingo bacteriales;Chitinophagaceae;Sediminibacterium;Sediminibacterium salmoneum NBRC 103935 0.025095 0.022915
SPKER >FJ230937.1.1465_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Alcaligenaceae;Achromobacter;uncultured bacterium 0.022918 0.021587
SPKER >DQ337037.1.1492_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Limnohabitans;uncultured bacterium 0.018802 0.017423
SPKER >HQ011601.1.1208_Bacteria;Proteobacteria;Betaproteobacteria;Methylophi lales;Methylophilaceae;Methylophilus;uncultured bacterium 0.016582 0.023566
SPKER >HQ592576.1.1490_Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Oxalobacteraceae;Massilia;uncultured bacterium 0.016219 0.016809
SPKER >BAUS01000005.232.1750_Bacteria;Proteobacteria;Betaproteobacteria;Met hylophilales;Methylophilaceae;Methylophilus;Methylophilus sp. OH31 0.014377 0.023272
SPKER >JN868908.1.1476_Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomo nadales;Sphingomonadaceae;Novosphingobium;uncultured bacterium 0.012791 0.012032
SPKER >JQ684487.1.1487_Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales ;Rhizobiaceae;Rhizobium;uncultured Sphingobium sp. 0.011509 0.010767
SPKER >GQ264220.1.1474_Bacteria;Proteobacteria;Betaproteobacteria;TRA3-20;uncultured bacterium 0.010951 0.009307
SPKER >HQ397490.1.1444_Bacteria;Proteobacteria;Alphaproteobacteria;Sphingom onadales;Sphingomonadaceae;Sphingomonas;uncultured bacterium 0.009231 0.008779
SPKER >JX219400.1.1436_Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; Bradyrhizobiaceae;Bosea;Starkeya sp. SL25 0.009213 0.008967
SPKER >EU887791.1.1495_Bacteria;Proteobacteria;Gammaproteobacteria;Oceanos pirillales;Oceanospirillaceae;Pseudospirillum;uncultured Cellvibrio sp. 0.009187 0.00863
SPKER >AY328708.1.1482_Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospiril lales;Rhodospirillales Incertae Sedis;Reyranella;uncultured bacterium 0.008175 0.007748
SPKER >JN087924.1.1518_Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcal es;Sandaracinaceae;uncultured;uncultured bacterium 0.006718 0.006332
SPKER >DQ354722.1.1491_Bacteria;Proteobacteria;Gammaproteobacteria;Oceanos pirillales;Oceanospirillaceae;Pseudospirillum;uncultured bacterium 0.006449 0.007264
SPKER >EU234308.1.1502_Bacteria;Proteobacteria;Gammaproteobacteria;Xanthom onadales;Solimonadaceae;uncultured;uncultured bacterium 0.002896 0.002722
Table SI. EMIRGE-assembled 16S rRNA gene sequences from metagenomic reads of eac 1
enrichment culture, and the top blastn hit from the SILVA 16S rRNA database (release 123). Prior
and normalized prior are metrics generated by EMIRGE to estimate relative abundance of each sequence. Nitrotoga sequences are in bold text.
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Hit Query Hit % ID Align- ment Length # Mis-matc hes Environment Reference
1 CP45_16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 100 1504 0 Estuary Sediment, China Li 2011
2 CP45_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.4 1504 9 Deglaciated Soil, Alaska, USA Sattin 2009
3 CP45_16S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.5 1497 7 Activated Sludge, China Unpublished
4 CP45_16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.3 1504 10 River Water, China Liu 2012
5 CP45_16S >KF533805 Candidatus Nitrotoga;uncultured bacterium 99.3 1498 9 Wastewater Treatment Plant, Denmark Ziegler 2016
1 LAW_16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 99.8 1504 3 Estuary Sediment, China Li 2011
2 LAW_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.3 1504 10 Deglaciated Soil, Alaska, USA Sattin 2009
3 LAW_16S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.3 1497 10 Activated Sludge, China Unpublished
4 LAW_16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.2 1504 11 River Water, China Liu 2012
5 LAW_16S >KF533805 Candidatus Nitrotoga;uncultured bacterium 99.3 1498 10 Wastewater Treatment Plant, Denmark Ziegler 2016
1 MKT_16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 99.9 1504 2 Estuary Sediment, China Li 2011
2 MKTJL6S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.5 1497 7 Activated Sludge, China Unpublished
3 MKT_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.3 1504 11 Deglaciated Soil, Alaska, USA Sattin 2009
4 MKT_16S >AB247475 Candidatus Nitrotoga;uncultured bacterium 99.3 1499 10 Activated Sludge, Japan Unpublished
5 MKT_16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.1 1504 12 River Water, China Liu 2012
1 SPKER_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.6 1504 6 Deglaciated Soil, Alaska, USA Sattin 2009
2 SPKER_16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 99.5 1504 7 Estuary Sediment, China Li 2011
3 SPKER_16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.5 1504 7 River Water, China Liu 2012
4 SPKER_16S >DQ839562 Candidatus Nitrotoga;Candidatus Nitrotoga arctica 99.7 1484 5 Permafrost Soil, Siberia, Russia Alawi 2007
5 SPKER_16S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.2 1497 12 Activated Sludge, China Unpublished
Table S2. Top five blastn hits of each SPAdes-assembled Nitrotoga 16S rRNA sequence against the SILVA 16S rRNA database (release 128).
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Full Text

PAGE 1

CULTIVATION AND GENOMIC SEQUENCING OF NOVEL NITRITE OXIDIZING BACTERIA by ANDREW MICH AE L BODDICKER B.A., University of Delaware, 2014 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Biology Program 2017

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ii This thesis for the Master of Science degree by Andrew Michael Boddicker has been approved for the Biology Program b y Annika C. Mosier, Chair Chris Miller Rebecca Ferrell Michael Greene Date: July 2 9 2017

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iii Boddicker, Andrew Michael (M.S. Biology Program) Cultivation and Genomic Sequencing of Novel Nitrite Oxidizing Bacteria Thesis directed by Assistant Pr ofessor Annika C. Mosier ABSTRACT Nitrification is a critical, rate limiting step in the removal of nitrogen pollution from freshwater systems. Nitrite oxidizing bacteria carry out an important regulatory function in the environment by converting nitrite t o nitrate, which is utilized by many microbes to facilitate nitrogen loss to the atmosphere. Recent research revealed that nitrite oxidation is carried out by a very diverse group of bacteria, with equ ally diverse metabolisms. F ew representatives have been cultured in the lab due to long incubations and heterotrophic contaminants. Here we describe the cultivation of novel freshwater Nitrotoga species, as well as the first reported Nitrotoga genomes. Four enrichment cultures were initiated from water column and sediment samples from Colorado rivers, each of which enriched a novel Nitrotoga species. Genomic DNA sequencing and assembly revealed highly conserved 16S rRNA gene sequence s but a surprisingly broad diversity among the rest of the genomes. A survey o f the core metabolism of each Nitrotoga species reveal ed genes for growth on organic carbon and growth at low oxygen concentrations, implicating potential to maintain function across a range of environments including biofilms, soil aggregates, and wastewater treatment plants. This work considerably expands our knowledge of Nitrotoga and improves our understanding of their role in the environment. Future efforts will focus on the res p onse of Nitrotoga to environmental change. The form and content of this abstract are approved. I recommend its publication. Approved: Annika C. Mosier

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iv ACKNOWLDEGEMENTS I would like to thank my advisor, Dr. Annika Mosier, for her phenomenal support over the course of this project; I really appreciate all that you do. Than k you to m y committee members, Dr. Chris Miller, for your extensive help with bioinformatics, and Dr. Rebecca Ferrell, for your great ideas and contributions. I am also grateful to Adrienne Narrowe, for her outstanding support and for teaching me how to be a better researcher. I have had the pleasure of working with several other students on this project and would like to thank Nick Deevers, Colin Beacom, Mike Kain, and especially Hannah Clark for starting these cultures. program without the support of friends from the M osier, Miller, and Roane lab s, as well as all of the other graduate students And a final thank you to my friends and family for their love and support.

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v TABLE OF CONTENTS CHAPTER I. INTRODUCTION ................................ ................................ ................................ ......................... 1 II. MATERIALS AND METHODS ................................ ................................ ................................ ...... 4 Culture Inoculation and Growth ................................ ................................ ........................... 4 Growth Curve ................................ ................................ ................................ ........................ 5 DNA Extraction ................................ ................................ ................................ ...................... 5 Sequencing ................................ ................................ ................................ ............................ 6 Metagenome and Nitrotoga Genome Assembly ................................ ................................ .. 6 Nitrotoga Genome Annotation ................................ ................................ ............................. 8 Nitrotoga Comparative Genomics ................................ ................................ ........................ 9 Nitrotoga Phylogenetics ................................ ................................ ................................ ....... 9 III. RESULTS AND DISCUSSION ................................ ................................ ................................ ...... 10 Enrichment & Nitrite Oxidation ................................ ................................ .......................... 10 Metagenome Assembly and Binning ................................ ................................ .................. 10 Nitrotoga Genome Assembly ................................ ................................ .............................. 12 Comparative Genomics ................................ ................................ ................................ ....... 14 Nitrotoga 16S rRNA Gene ................................ ................................ ................................ ... 16 Nitrotoga Carbon Metabolism ................................ ................................ ............................ 18 CO 2 Fixation ................................ ................................ ................................ ..................... 18 Organic Carbon Utilization ................................ ................................ .............................. 19 Nitrotoga Nitrogen Metabolism ................................ ................................ ......................... 20 Nitrite Oxidation ................................ ................................ ................................ ............. 20 Assimilatory and Dissimilatory Nitrogen Metabolism ................................ .................... 27 Nitrotoga Electron Transport for Energy Conservation ................................ ...................... 28 Nitrotoga Motility and Chemotaxis ................................ ................................ .................... 30 Nitrotoga Membrane Transport, Signaling, and Defense ................................ ................... 31 IV. CONCLUDING REMARKS ................................ ................................ ................................ .......... 33 REFERENCES ................................ ................................ ................................ ................................ ......... 35 A PPENDIX : Supplemental Materials ................................ ................................ ................................ ..... 42

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1 C HAPTER I I NTRODUCTION Ever increasing anthropogenic sources of nitrogen, such as fertilizer and wastewater, have led to environmental risks including eutrophication, acidification, and increased greenhouse gas em issions. Nitrite oxidizing bacteria (NOB) play a critical role in mitigating the harmful effects of nitrogen pollution by linking nitrogen sources to nitrogen removal processes. Specifically, nitrite pools from natural (e.g. ammonia oxidation) or anthropo genic (e.g. fertilizer) sources are converted into nitrate, which is then removed from the system as inert nitrogen gas via denitrification or anammox. Thus, understanding the physiology, metabolism, and environmental limits of NOB is important for contro lling and managing elevated nitrogen in contaminated ecosystems. Despite the environmental relevance of NOB, the group is understudied in part due to their slow growth in the lab and extended enrichment periods, taking as long as 12 years for isolation (Lebedeva et al. 2008) Assiduous cultivation efforts (Watson and Waterbury 1971; Watson et al. 1986; Alawi et al. 2007; Sorokin et al. 2012; Daims et al. 2015; van Kessel et al. 2015) as well as single cell (Ngugi et a l. 2015) and metagenomic (Pinto et al. 2015) sequencing studies are beginning to illuminate the diversity of NOB. NOB have been found in four phyla and seven different genera three of which were dis covered within the last decade : Nitrotoga Nitrolancea and Can didatus Nitromaritima (Alawi et al. 2007; Sorokin et al. 2012; Ngugi et al. 2015) Members of the Nitrobacter and Nitrospira genera have b een relatively well studied both in the environment and in culture, but our understanding of nitrite oxidation by members of other genera is deficient. NOB are associated with not only a wide range of phylogen e tic diversity, but also physiologic versatilit y, including complete ammonia oxid ation (comammox within the Nitrospira ) mixotrophic growth with organic

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2 carbon, differences in electron transport, and features that enable niche development in specific habitats ( Starkenburg et al. 2008; Lcker et al. 2010; Lcker et al. 2013; Daims et al. 2015; van Kessel et al. 2015; Ngugi et al. 2015; Daims et al. 2016) Of the currently described NOB, Nitrotoga have recently become an area of focus for nitrification research due to their ubiquity in natural and engineered environments, including wastewater treatment plants where they may play an important functional rol e (Lcker et al. 2015) Recent m olecular surveys have identified Nitrotoga like sequences in a surprisingly wide range of habitats, includ ing: glacial soils (Sattin et al. 2009; Schmidt et al. 2009; Pradhan et al. 2010; Srinivas et al. 2011) an underground cave (Chen et al. 2009) a freshwater seep (Roden et al. 2012) a dr inking water filter (White et al. 2012) a subglacial Antarctic lake (Christner et al. 2014) Yellow Sea seawater (Na et al. 2011) salt marsh sediments (Martiny et al. 2011) rivers (Fan et al. 2016) antibiotic impacted rivers (Li et al. 2011) and various wastewater treatment facilities (Bereschenko et al. 2010; Karkman et al. 2011; Lcker et al. 2015; Saunders et al. 2015; Ziegler et al. 2016; Yang et al. 2016) T he relative proport ion of Nitrotoga like sequences in several wastewater treatment facilities and a subglacial lake were as high as 2 13% of the total estimated bacterial community, and were occasionally the only observed NOB (Christner et al. 2014; Lcker et al. 2015; Saunders et al. 2015) The distribution and relative abundance of Nitrotoga species across the globe indicate that these organisms likely play a critical role in global nitrogen cycling in a diverse range of environments; however few studies have attempted to characterize their environmental im pact Candidatus Nitrotoga arctica, enriched from permafrost affected soils in 2007, was the first identified member of the novel genus Nitrotoga (Alawi et al. 2007) T hree other members of the same genus have since been enriched from a wastewater treatment pla nt aquaculture system and coastal sand (Alawi et al. 2009; Hpeden et al. 2016; Ishii et al. 2017) These Nitrotoga enrichments

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3 have begun to be characterized physiologically, but there are no confirmed isolates and no avai lable genomes to date. Without genomic data, our understanding of potential Nitrotoga metabolic versatility and niche adaptation in different environments is lacking. The four previously reported Nitrotoga en richments were all derived from cold environments, and were enriched at temperatures between 4C and 17 C to improve selection. Nitrotoga sp. HW29 retain ed about 40% of its nitrite oxidizing capabilities at 10C but had an optimal temperature for growth of 22C (Hpeden et al. 2016) The upper limits for growth among the four enrichments (as measured in culture) were 25 2 9C. Nitrotoga sp. HW29 showed gro wth at pH as low as 5.7, and slightly acidic media improved purification of the enrichment (Hpeden et al. 2016) Ca. Nitrotoga arctica has an intermediate nitrite affinity when compared to Nitrobacter and Nitrospira NOB and is adapted to low nitrite concentrations (0.3 mM) (Nowka et al. 2015) Here, we describe the draft genome sequences of four novel enrichments of distinct Nitrotoga species. Each organism contains copies of all essential nitrite oxidation genes, as well as complete electron transport chai ns, and capabilities for inorganic carbon fixation and organic carbon degradation. The possession of high affinity terminal oxidases, as well as chemotaxic and oxygen binding molecules, indicate that these Nitrotoga species are likely able to survive in lo w oxygen environments These findings extend our understanding of freshwater nitrite oxidation, and form the basis for further experimental work aimed at testing genomic predictions in culture and in the environment.

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4 CHAPTER II MATERIALS AND METHODS Cult ure Inoculation and Growth Enrichment cultures were grown in Freshwater Nitrite Oxidizer Medium (FNOM) with a nitrite co FNOM wa s prepared by mixing 1 g NaCl, 0.4 g MgCl 2 6H 2 O, 0.1 g CaCl 2 2H 2 O, 0.5 g KCl, 100 L 10X vitamin solution (Balch et al. 1979) 1 mL 1M NaHCO 3 and 300 L of 1M NaNO 2 per liter. The pH of the media was lowered to 7.0 using 10% HCl, and it was then autoclaved. After autoclaving, 10 mL of separately autoclaved 4 g/L KH 2 PO 4 and 1 mL trace metals solution (Biebl and Pfennig 1978) was sterilely added to the media before storing at 4C in the dark. S ediment s amples (n=2) were taken in February of 2015 from two locations of Cherry Creek in downtown Denver, CO ( samples MKT and LAW) Sediment samples were taken aseptically using a cut off sterile syringe, and returned to the lab on ice. The same day, 0.5 cm of sediment (~3 mL) was well mixed with 10 mL of sterile FNOM and then 1 mL of the sediment mixture was transfe rred to 100 mL of sterile media for incubation at room temperature in the dark Water column samples (n=2) were taken in May of 2015 from two rivers in a more agri culturally dominated region near Greeley, CO, about 100 km North of Denver CO ( samples CP45 from the Cache La Poudre River and SPKER from the South Platte River ) River water was kept on ice in the field and stored at 4C upon return to the lab. After fiv e days, 10 mL of water from each site was transferred to sterile FNOM and allowed to incubate at room temperature in the dark. Growth of enrichment cu ltures was regularly monitored using a Gr i e ss nitrite color reagent (Griess Romijn van Eck 1966) composed of 0.5 g sulfanilamide, 0.05 g N (1 naphthyl) ethylenediamine dihydrochloride, 5 mL 85% phosphoric acid, and Mill iQ water to a final volume of

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5 50 mL. The solution wa Nitrite color reagent was mixed with the culture separately at a 1:1 or 1:10 ratio. Standard enrichment cultures were transferred to new media approximately every two weeks. Enrichment was enhanced by serial dilution, rapid transfer as soon as nitrite was starting to disappear, and low volume trans fers (as low as 0.1% inoculum). Enrichmen t for Nitrotoga spp. was later enhanced by moving a subset of cultures to grow at 10C and media pH was dropped to 6 .0 before autoclaving (Alawi et al. 2007; Alawi et al. 2009; Hpeden et al. 2016) C ulture stocks were frozen in 10% DMSO as described by Vekeman et. al ( 2013) but without TSB addition and only one wash step to reduce loss of cells. Additional aliquots were fixed in 4% paraformaldehyde for future fluor e s cence in situ hybridization experiments. Growth Curve Triplicate cultures of each culture line (CP45, LAW, MKT, and SPKER) were inoculated with inoculum into 100 mL fresh media to mea sure nitrite oxidation rates. S amples of each culture were collected at regular intervals for nitrite measurements. Equal volumes of fresh Griess nitrite color reagent and culture were mixed, and the optical density was measured at 540, 545, and 550 nm using a BioTek Synergy HT p late r eader (BioTek, Winooski, VT). Mean max OD was used to calculate nitrite concentrations based on c o mparison with a standard curve of sterile medi a ranging from 0 were made in triplicate. DNA Extraction Liquid cultures w ere filtered through a 0.2 ( Pall, New York, NY) then removed with sterile forceps cut with a sterile scalpel and aseptically placed into a Lysing Matrix E Bead Beating Tube ( MP Biomedicals, Santa Ana, CA) with

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6 mM EDTA, 400 mM NaCl, 50 mM Tris ( The solution was vortexed briefly before bead beating in a FastPrep 24 5G reciprocating homogenize r ( MP Biomedicals, Santa Ana, CA) at 5 m/s for 3 0 seconds. The samples were incubated at 99C in a dry bath for 1 3 minutes before adding The samples were then incubated for at least 3 hours (up to overnight) in a rotating hybridization oven at 55C. Cold 100% ethanol ( 50 ) was added to th e sample in the Lysing Matrix E Bead Beating Tube. DNA was then purified using the (Qiagen, Hilden, Germany) I solated DNA was quantified with a Qubit fluorometer ( Thermo Fisher, Waltham, MA) Sequencing Extracted DNA was sequ enced at the University of Color ado Anschutz Medical Campus Genomics Core on an Illumina HiSeq 2500 using V4 chemistry ( Illumina, San Diego, CA) with 2x125 bp paired end reads. DNA was sheared using a Covaris S220 (Covaris, Woburn, MA) and l ibraries were prepped with an insert size of 400 bp using an Ovation Ultralow System V2 (No. 0344) kit ( Nugen, San Carlos, CA) Metagenome and Nitrotoga Genome Assembly Sequencing adapters were removed and reads were quality trimmed and filtered using BBDuk ( http://jgi.doe.gov/data and tools/bbtools/ ). Reads with adapter overlaps of at least 8 bp (mink=8) with one al lowed mismatch (hdist=1) were trimmed, as well as bases under a Phred score threshold of 20 (qtrim=20) and a minimum final read length of 50 bp (minlength=50). Additional bases beyond 125 bp were removed (ftm=5), while both reads in a mate pair were trimme d (tpe), and merged mate pairs were used to trim adapters based on overlap (tbo). Read distributions were

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7 manually checked for quality using FASTQC ( https://www.bioinformatics.ba braham.ac.uk/projects/ fastqc/ ) before and after trimming and filtering 16S rRNA genes were assembled f rom the t rimm ed and filtered reads using the EMIRGE platform (Miller et al. 201 1) T axonomy was assigned via a blastn (BLAST+ 2.6.0) (Camacho et al. 2009) search against the SILVA 16S rRNA database (release 123) The assembled 16S rRNA gene sequences later helped to confirm binning and taxonomic assignments of assembled genomes. Metagenomi c reads from each culture were assembled separately using MEGAHIT (Li et al. 2014) with kmer sizes ranging from 31 to 121, in step s of 10. Reads were mapped to contigs using BBMap ( http://jgi.doe.gov/data and t ools/bbtools/ ) and then binned using MetaBAT ( -verysensitive B20) (Kang et al. 2015) Bin completeness and contamination estimates were calculated using (Parks et al. 2015) pipeline version 2.1.0 (Eren et al. 2015) but instead of submitting multip le metagenome samples, each bin was treated as its own and combine bins when necessary Combined bins were re analyzed with CheckM to determine if completeness estimates increased while contamination estima tes stayed similar. Genes were called within each bin using Prodigal (Hyatt et al. 2010) and blastp was used to find the best hit for each gene from bins of inte rest against the UniRef90 database (release 2016_11) (Suzek et al. 2014) Individual contigs with suspect taxonomic results were scrutinized and removed upon later reassembly (see below) Reads were mapped back to the contigs of all individual bins using BBSplit ( http://jgi.doe.gov/data and tools/bbtools/) and were used for reassembly with SPAdes 3.9.0 (Bankevich et al. 2012) SPAdes was run under the careful setting with MEGAHIT contigs given as and kmer sizes from 31 to 121, in steps of 10. Assembly graphs were visualized

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8 using Bandage (Wick et al. 2015) to check for suspected connections between contigs, as well as to identi fy potential outlier contigs. Metagenome assembled contigs that were not incorporated into larg er SPAdes assembled contigs, or were not contiguous with other contigs were additionally scrutinized. If contigs had dissimilar best Uni Ref90 hits, inconsistent blastx hits against the NCBI nr database, and were not found to be present within contigs of o ther Nitrotoga genomes (from this study), they were removed from the bin before further reassembly. An iterative reassembly process followed as above while adding reads that mapped to suspected contigs with 16S rRNA gene sequences, which rarely binned properly. To accomplish this, all metagenome assembled contigs were searched against the SILVA 16S rRNA database (release 128) (Quast et al. 2013) and those with alignment lengths of >300 bp (or 189 bp for LAW due to Nitrotoga being the best hit for a fragment of that size) that did not fall into appropriate bins, were added to the assembly. The internal BLAST function of Bandage allowed visualization of each 16S rRNA gene contig after reassembly, and only contigs that assemble d into the contiguous genome were kept. In all cases, only the Nitrotoga 16S rRNA gene contigs assembled with the rest of the genome. A similar process was followed for adding contigs with nxr gene sequences to the assembly; however this occurred after fin al annotation. After each iterative assembly, CheckM (Parks et al. 2015) w as run to establish any changes in completeness or contamination, and read mappings were visualized using Tablet (Miln e et al. 2013) to identify potential misassemblies. Nitrotoga Genome Annotation Final reassemblies were filtered to remove contigs <2 kb, all of which had very low and uneven coverage estimates. The Nitrotoga g enomes were aligned and contigs reordered with

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9 progressiveMauve (Darling et al. 2010) using the Nitrotoga sp. from the MKT culture as a reference due to its long contig length and simple assembly graph. Genomes were submitt ed to the DOE JGI Microbial Genome Annotation Pipeline (MGAP) (Huntemann et al. 2015) for final contig trimming of ambiguous and low complexity sequences, and functional gene annotation. KEGG pathway maps (Kanehisa and Goto 2000) were used to e valuate the core metabolism of Nitrotoga P redicted sig nal peptides and transmembrane regions of annotated pr oteins were evaluated using SignalP (Petersen et al. 2011) and TMHMM (Krogh et al. 2001) Nitrotoga Comparative Genomics Average nucleotide identity (ANI) was calculate feature (Eren et al. 2015) was used to cluster coding sequences (CDS) from each Nitrotoga of genes shared by all four genomes. Unique genes found in each genome were identified, as well as those shared among two or three of the genomes. Nitrotoga Phylogenetics Gene and protein sequences of interest were iteratively aligned using MUSCLE (Edgar 2004) or MAFFT (Katoh et al. 2002) and then manually edited Re ference sequences were taken from close relatives and other NOB for 16S rRNA sequence alignment (1,496 bp) as well as other m embers of the Type II DMSO reductase family for amino acid sequence alignment of nitrite oxidoreductase ( 1,344 amino acids) Phylogenetic trees were generated using FastTree (Price et al. 2010) with default param e ters including 1,000 resamples without branch length reoptimization. All phyl ogenetic analyses were run using Geneious version 8.1.8

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10 CHAPTER III RESULTS AND DISCUSSION Enrichment and Nitrite Oxidation Four enrichment cultures were initiated from freshwater sediment and water column samples of Colorado rivers in 2015 Two samples (LAW and MKT) were enriched from the sediment of a heavily contaminated urban creek, and two (CP45 and SPKER) were enriched from the water column of rivers dominated by agriculture and livestock land us e The cultures were enriched for 17 (CP45 and SPKER) or 20 (LAW and MKT) months via serial dilution and rapid transfer to new media before DNA was extracted for sequencing. Nitrite oxidation rates were determined by measuring nitrite consumption over the course of 22 days (rates calculated during exp onential growth). The nitrite oxidation rates of each culture were : 2 /day (SPKER) 2 2 /day (CP45), and 141.6 2 / day (MKT). 2 /day which was similar to values re corded for Ca. Nitrotoga arctica (Nowka et al. 2015) Metagenome Assembly and Binning Evidence from EMIRGE 16S rRNA gene assembly (Miller e t al. 2011) suggested there may be overlap in taxonomy among the four samples based on simila r blast hits among cultures to similar taxa. H owever (Eren et al. 2015) suggested there was very little overlap in terms of genomic diversity between samples, including the four Nitrotoga genomes (Table S1 and Fig S1) Additional attempts at a co assembly using all reads from all cultures, or all Nitrotoga reads, were unsuccessful as each culture contains unique organisms (Fig. S1)

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11 Figure 1. Nitrite oxidation by Nitrotoga enrichment cultures over time Nitrite concentration was quantified colorimetrica lly. Each enrichment culture was inoculated in triplicate and logarithmic dec lines in nitrite concentration we re presumed to represent the logarithmic growth phase of the nitrite oxidizing Nitrotoga species. Error bars show the standard deviation of each m easurement. A fter curation of the assembly, the samples contained 8 (CP45), 14 (MKT), 23 (LAW), and 32 (SPKER) genome bins and the Nitrotoga genome bins were the most abundant, or one of the most abundant, organisms based on genome coverage The majority (47/77) of these bins were determined to be near complete (>90% based on CheckM estimates). The remaining 30 bins had <90% completeness or had high contamination estimates which necessitate s further refinement before analysis. The CheckM lineage workflow w as used, which assigns a taxonomic lineage to the genome bin to determine a set of marker genes to use in completeness and contamination estimates ; therefore incomplete bins may be assigned root taxonomy (e.g. k__Bacteria with 104 markers) with fewer marke r genes than specific lineag es (e.g. c__Betaproteobacteria UID3959 with

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12 419 markers) inflating or deflating completeness and contamination estimates. Besides Nitrotoga each enrichment culture consisted almost exclusively of Proteobacteria, including at l east one highly abundant Pseudomonas spp. as well as a common Methylotenera spp. and several members of the Comamonadaceae family. One genome bin from the SPKER metagenome likely belongs to a protozoan in the Neobodonida order that may prey on bacteria T he Nitrotoga bins were called as Betaproteobacteria Many of the preliminarily called genes (~50%) from these contigs had best hits to genomes within the Gallionellaceae family when compared using blastp against the UniRef90 database (Suzek et al. 2014) which includes the iron oxidizing Gallionellea and Sideroxydans genera. Nitrotoga Genome Assembly Each enrichment culture was found to contain a unique Nitrotoga spp., which w as tentatively named base d on sampling location: Nitrotoga sp. CP45, Nitrotoga sp. LAW, Nitrotoga sp. MKT, and Nitrotoga sp. SPKER. T he enrichment of Nitrotoga from freshwater has not previously been documented, and no Nitrotoga genomes are publically available Thus, the genomic characteristics described here will vastly improve our understanding of Nitrotoga and how they may behave in natural and engineered environments. The Nitrotoga genomes ranged in size from 2. 707 2.9 82 Mbp, with 23 5 9 contigs and GC content between 47.5 % an d 48.8% (Table 1) Assembly N50 statistics (69.7 214.8 kbp) were associated with the number of total contigs from each genome (i.e. genomes with fewer contigs typically had higher N50 values). The number of coding sequences ranged from 2,574 2,858 with 36 39 tRNAs encoding all twenty amino acids. CheckM estimates indicated that the four Nitrotoga genomes were near complete, based on comparisons to a collection of 419 single copy gene markers that are conserved within the

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13 Betaproteobacteria (UID3959). Nitrotoga sp. LAW, MKT, and SPKER genomes were 98. 2 % complete, while the CP45 genome was 97.0% complete due to the loss of three markers that were present in contigs <2 kb which were removed before annotation (PF00731.15 AIR carboxylase; PF01259.13 Phospho ribosylaminoimidazolesuccinocarboxamide synthase; and TIGR02392 alternative sigma factor RpoH) All four genomes were missing the same five marker gene s (TIGR01745 aspartate semialdehyde dehydrogenase ; TIGR01574 tRNA i(6) A37 thiotransferase enzyme MiaB; P F03618.9 Kinase PPPase ; TIGR01161 phosphoribosylaminoimidazole carboxylase, ATPase subunit ; and PF13603.1 Leucyl tRNA synthetase, Domain 2 ) A manual search of each HMM using HMMER (Eddy 2011) reveal ed strong (evalue <1e 41) hits to four of the five missing marker genes in all four genomes. Two markers (TIGR0 1745 and PF13603.1) had Nitrotoga genome annotations from the JGI pipeline that were identical to the HMM Two other markers had similar annotations and strong hits (TIGR01574 and TIGR01161). The addition of these four marker genes (and three removed markers from CP45) would improve completeness estimates t o 99.8% complete. The fifth marker gene (PF03618.9) had no hits from the JGI annotated genomes, and was not found on a contig with suitable coverage in the unbinned contigs from any of the four samples. Homologs to this marker gene are found in the genomes of the closest sequenced relatives: Gallionella a cididurans ShG14 8, Gallionella capsiferriformans ES 2, and Sideroxydans lithotrophicus ES 1, indicating that this gene may indeed be missing from the assembly.

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14 Table 1. Assembly statistics overview for Ni trotoga genomes. SPAdes was used for final assemblies and annotations were completed using the DOE JGI Microbial Genome Annotation Pipeline. The NXR containing contig was manually annotated and added to the assembly statistics for each genome The minimum contig sizes were 2,505 bp (CP45), 4,389 bp (LAW), 4,076 bp (MKT), and 2,458 bp (SPKER). The Nitrotoga genomes were estimated to contain 0.24 0.30% contamination. Specifically, t hree of the genomes (CP45, LAW, and MKT) had duplications of the same two markers (PF09976.4 Tetratricopeptide repeat like domain ; PF08340.6 d omain of unknown function 1732 ) while Nitrotoga sp. SPKER only ha d a duplicate of PF08340.6 One of the copies of each duplicated marker gene had a drastically reduced protein length and weak HMM hit, so they may represent false positives for gene detection. None of the published Gallionellaceae genomes showed duplications of these genes. Nitrotoga Comparative Genomics Average nucleotide identity (ANI) was calculated pairwise between the four Nitrotoga geno mes and two most closely related genomes of iron oxidizers from within the Gallionellaceae family: Sideroxydans lithotrophicus ES 1 and Gallionella capsiferriformans E S 2 (Table 2) ANI values between the Nitrotoga genomes and the iron oxidizers ranged from 71.3% to 72.6% identity, indicating a vast phylogenetic distance even from the closest related sequenced genomes Therefore the iron oxidizer genomes could not be us ed as a close reference in Nitrotoga genome assembly, and supports the classification of Nitrotoga as a novel candidate genus (Alawi et al. 2007) The Nitrotoga sp. CP45, LAW, and MKT genomes were all closely related to each other with ANI values ranging from 93. 7% to 94.4 %. The Nitrotoga sp. SPKER genome, however, was much more distinct,

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15 with ANI values ranging from 87. 1 % to 87.4% when compared to the other three Nitrotoga genomes. These values are indicative of species level differences between each culture (Richter and Rossell Mra 2009) The variance in genome content when compared to the conservation of 16S rRNA gene sequences (see below) is also noteworthy, as studies that cluster 16S rRNA sequences alone may under estimate species diversity of the Nitrotoga ne was run on the four Nitrotoga genomes to identify conserved coding sequences (Eren et al. 2015) (Fig. S2). Proteins we re clustered using an MC L algorithm to identify homolog s at the amino acid level between genomes. Overall, 1,794 protein meaning all four genomes possess ed these coding sequences. Each genome also encoded unique coding seque nces not found in any other Nitrotoga genome (CP45: 365 protein clusters, LAW: 348 protein clusters, MKT: 293 protein clusters, SPKER: 626 protein clusters), and 568 protein clusters were found to be shared between two or three genomes only.

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16 Table 2. Pairwise average nucleotide identity (ANI) comparisons between enriched Nitrotoga genomes and the two closest available genomes calc ompare G enomes feature These values represent species level differences between Nitrotoga genomes. Nitrotoga 16S rRNA Gene The Nitrotoga genomes contained surprisingly conserved 16S rRNA gene sequences when compared to the genome sequence divergence. The Nitrotoga sp. CP45 and MKT 16S rRNA gene sequences were 99.87% identical with only two mis matches across the entire 1544bp gene, while the most distantly related were MKT and SPKER at 99.42% identity and nine mismatches. The 16S rRNA sequences were also closely related (>88% identity, average 96% identity) to publically available Nitrotoga sequ ences from the SILVA database (release 128) (Quast et al. 2013) environmental gene surveys (from soil, sediment, river water, and wastewater samples) except for one SPKER hit to the original Candidatus Nitrotoga arctica clon e (Table S2). All four 16S rRNA

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17 sequences grouped phylogenetically with other Nitrotoga sequences within the B etaproteobacteria and were divergent from nitrite oxidizers in other phyla (Fig. 2). Figure 2. 16S rRNA sequence phylogenetic tree of NOB and close relatives aligned across 1,496 bp. Members of all seven nitrite oxidizer genera are present, and our 16S rRNA sequences are most similar to other Nitrotoga sequences from enrichment cultures within the B etaproteobacteria. Other nitrite oxidizers are found in the Alphaproteobacteria ( Nitrobacter ), Gammaproteobacteria ( Nitrococcus and Thiocapsa ), Nitrospinae ( Nitrospina and Candidatus Nitromaritima), Chloroflexi ( Nitrolancea ), and Nitrospirae ( Nitrospira ). Nodes with FastTree support values >50% are shown with a blue circle. The 5S, 23S, and 16S rRNA genes all assembled together along with two tRNA genes in each genome However, these genes all had approximately double the coverage of the surrounding contig when reads were mapped back, likely representing a repeat. The operon was split between multiple contigs in the LAW and SPKER genomes resulting in inflated numbers of predicted rRNA genes by MGAP (Table 1), while the same sequence was found in the middle o f large contigs for CP45 and MKT.

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18 Nitrotoga Carbon Metabolism CO 2 Fixation All described NOB possess one of two inorganic carbon fixation pathways : the reverse tricarboxylic acid cycle (rTCA) has been found either biochemically or via genome surveys in the Nitrospina Nitrospira and Candidatus Nitromaritima (Lcker et al. 2010; Lcker et al. 2013; Ngugi et al. 2015) ; and the Calvin Benson cycle dominates in the Nitrobacter Nitrococcus and Nitrolancea ( Starkenburg et al. 2008; Sorokin et al. 2012) Nitrotoga enrichment cultures were g rown in media containing no organic carbon suggesting they also must have a method of fixing inorganic carbon. Indeed, all four of the Nitrotoga genomes were found to contain genes coding for 12 of 13 enzymes used in the Calvin Benson cycle to fix carbon d ioxide. The missing gene in all four genomes was sedoheptulose bisphosphatase, which catalyzes the hydrolysis of sedoheptulose 1,7 bisphosphate to sedoheptulose 7 phosphate. Genes coding for this enzyme were also missing from the genomes of another NOB, Ni trobacter winogradskyi, as well as the ammonia oxidizing bacteria Nitrosomonas europaea and Nitrosospira multiformis (Chain et al. 2003; Starkenburg et al. 2006; Norton et al. 2008) Fructose 1,6 b isphosphatase, which typically plays a role in gluconeogenesis and was present in all four Nitrotoga genomes, may fill this role in these genomes, as well as in other organisms (Yoo and Bowien 1995; Wei et al. 2004) One gene encoding sedoheptulokinase, a bidirectional phosphorylating accessory enzyme phosphate) to the Calvin Benson cyc le, was missing from all four Nitrotoga genomes. Additionally, the SPKER genome was missing genes encoding for xylose 5 phosphate/fructose 6 phosphate phosphoketolase, which play a role in the Calvin Benson cycle, but are not critical to carbon fixation. T he fructose 6 phosphate phosphoketolase catalyzes the same re action as transketolase, which wa s present in all four Nitrotoga genomes, and the xylose 5

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19 phophate regenerates glyceraldhyde 3P from xylulose 5P, which can also be accomplished via other enzymes of the Calvin Benson cycle. All four Nitrotoga genomes contained two copies of the ribulose( 5 ) phosphate 3 epimerase enzyme, and SPKER contained two copies of transketolase genes, both of which play roles in the Calvin Benson cycle and the pentose phosp hate pathway but other key genes were missing from the pentose phosphate pathway The CP45, LAW, and MKT genomes all had two copies of both large and small ribulose 1,5 bisphosphate carboxylase subunits (RuBisCO ), the key enzyme for carbon dioxide fixa tion, while the SPKER genome had a single copy. Organic Carbon Utilization In addition to carbon fixation, all four Nitrotoga genomes had genes encoding proteins for complete glycolysis and pentose phosphate pathways, but not gluconeogenesis. A phosphoen olpyruvate carboxylase gene was missing from all four genomes, which catalyzes the first step of gluconeogenesis (oxaloacetate phosphoenolpyruvate). All other enzymes in gluconeogenesis are the same as those used in glycolysis, except for fructose 1,6 bi sphosphatase, which was present in all four genomes but may play a role in carbon fixation rather than gluconeogenesis (see above). Additionally, genes for glucose transport enzymes were missing from all four Nitrotoga genomes. All four Nitrotoga genomes h ad genes encoding enzymes that generate acetyl CoA from pyruvate, as well as an acetyl CoA synthetase, which produces acetyl CoA directly from acetate. All Nitrotoga genomes contained the full suite of genes for the TCA cycle, but w ere missing both key enzymes of the reverse TCA cycle for CO 2 fixation, ATP citrate lyase and 2 oxoglutarate:ferredoxin oxidoreductase, as used by Nitrospira defluvii (Lcker et al. 2010) These co mplete pathways

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20 suggest that Nitrotoga species are likely capable of heterotrophic growth on organic carbon which has also been reported in a wide variety of other NOB (Daims et al. 2016) Nitrotoga Nitrogen Metabolism Nitrite Oxidation All described NOB genomes (from the Nitrobacter Nitrospina Nitrococcus Nitrospira Nitrolancea and Candidatus Nitromaritima genera) contain genes necessary to form a functional nitrite oxidoreductase (NXR), a three subunit enzyme ( nxrA alpha subunit, nxrB beta subunit, nxrC gamma subunit) that oxidizes nitrite to nitrate, stripping two electron s for use in energy conservation. NXR belongs to the Type II DMSO reductase family of molybdenum enzymes (Lcker et al. 2010; Daims et al. 2016) in which the active site of the a lpha subunit contains a molybdenum bismolydopterin guanine dinucleotide (Mo bisMGD) motif, as well as one iron sulfur cluster (Magalon et al. 2011; Hille et al. 2014; Daims et al. 2016) The beta subunit contains four iron sulfur clusters for electron conduction away from the alpha subunit towards the membrane integral gamma subunit, w hich is less well understood and is thought to contain up to two heme groups that may be involved in electron transfer to a cytochrome c for delivery to the electron transport chain (Lcker et al. 2010; Magalon et al. 2011; Daims et al. 2016) The most prominent difference in the NXRs of NOB is the orientation of the enzyme in relation to the cell membrane. Members of the Nitrobacter Nitrococcus and Nitrola ncea have been shown to possess cytoplasmic facing NXR (Spieck et al. 1996; Starkenburg et al. 2006; Sorokin et al. 2012) while members of the Nitrospira and Nitrospina have been shown to have periplasmic facing NXR (Spieck et al. 1998; Lcker et al. 2010; Lcker et al. 2013; Ko ch et al. 2015) Candidatus Nitromaritima was presumed to have a periplasmic NXR due to predicted signal peptides on the alpha subunit for excretion via the twin arginine translocation (Tat) pathway (Martinez espinosa et

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21 al. 2007; Sargent 2007; Ngugi et al. 2015) Nitrotoga we re also predicted to have a periplasmic facing NXR (Nowka et al. 2015) Oxidation kinetics associated with NXR orientation have been established (Nowka e t al. 2015) and have implications in ecological niche formation, as bacteria with cytoplasmic facing NXR typically dominate in relatively high nitrite environments over their periplasmic facing counterparts In three of the Nitrotoga genome s (CP45, LAW and MKT) a single 8.7 kb contig had strong hits to available T ype II DMSO HMMs for the alpha (e value 1e 78) beta (e value 1e 77) and gamma subunits (e value 1e 39) (TIGR03477 TIGR03479) as well as a designated chaperone n xrD (e value 1e 29) (TIGR03 482) that is similar to TorD used in molybdenum cofactor assembly and protein folding (Magalon et al. 2011; Lcker et al. 2013; Hille et al. 2014; Ngugi et al. 2015) Together, these form an n xrDCBA operon. E ach contig also encoded a putative 70 a mino a cid protein of unknown function, and a 341 a mino a cid protein with conserved domains related to iron transfer P loop NTPases, which are required for cytosol ic Fe S cluster assembly ( factor NBP35) both located upstream of the nxr genes CP45 also had two additional hypothetical proteins, one on each end of the contig. Reads mapped from each end of the NXR contig (in the LAW and MKT genomes) to two other cont igs within the assembly, likely representing a repeated region with two distinct locations within each genome. The CP45 NXR contig however, did not have mapped reads leading to other contigs, and the ends of the contig had much lower read coverage than th e interior, indicating that more sequence surrounding this region may be missing The CP45, LAW, and MKT NXR containing contigs had similar GC content and approximately double coverage estimates when compared to the rest of their respective genomes, supporting the presence of gene duplications with very high

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22 similarity. In all three cases, mapped reads did not indicate any single nucleotide polymorphisms (SNPs) present in the contig, likely representing two identical copies. The SPKER genome did not have a single contig corresponding to all NXR genes, but rather the genes were scattered across several contigs The SPKER NXR contig s assembled with the rest of the genome, with connections to four other contigs, but coverage estimates indicate d this geno me may have three copies of the nxr genes. The four genes ( n xr DCBA ) assembled in the same order as all other genomes, but the contig was lacking copies of the two other genes (FeS cluster assembly factor NBP35 and 70 a mino a cid hypothetical protein) found in the same contig from all three other Nitrotoga genomes. A copy of the FeS cluster assembling factor NBP35 gene was found on a connected contig, but only at single coverage. Mapped reads to this contig also indicate d that there may be two different nxrA sequences due to the presence of SNPs. Previous reassemblies using contigs below the coverage threshold for this genome ass embled two different copies of nxrA one with single coverage and one with double coverage H owever the final reassembly resulted in only one nxrA sequence as there was not enough divergence in read mapping to isolate different copies The nxrDCB genes always assembled at approximately triple coverage when compared to the rest of the genome. The putative NXR alpha subunit s from CP45, LAW, and MKT had a Tat signal peptide on the N terminal as predicted by the SignalP 4.1 online server (Petersen et al. 2011) which supports the excretion of this enzyme to the periplasm (Sargent 2007) The SPKER NXR alpha subunit wa s lacking a Tat signal peptide, although an alignment of the four copies show ed that the first 14 amino acids may be missing, as the entire peptide wa s otherwise aligned without gaps (Fig. 3) The putative beta subunit wa s lacking a si gnal peptide, but may be previously in other NOB (Martinez espinosa et al. 200 7; Lcker et al. 2010; Pester et al. 2013)

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23 The putative gamma subunit of each genome ha d an N terminal signal peptide which was previously observed in the genomes of Nitrospina gracilis and Candidatus Nitromaritima spp. (Lcker et al. 2013; Ngugi et al. 2015) However, in both cases the predicted signal peptide cleavage site was located in the middle of a predicted transmembrane region and previous studies had confirmed the membrane association of the NXR complex in other NOB genera indicating that these gamma subunits were not fully translocated into the periplasm (Lcker et al. 2010) In the case of Nitrotoga the putative nxrC gene wa s located directly next to the alpha and beta subunit genes, but did not contain any predicted transmembrane regions (by InterProScan). This subunit is predicted to belong to the heme binding members of the Type II DMSO reductase family, however it is more similar to the soluble gamma subunit of ethylbenzene dehydrogenase than the membrane bound subunit of NXR (Kloer et al. 2006; Magalon et al. 2011) Much less is known about the structure and function of the N xrC subunits as gene copies even within the same gen ome can vary in size, identity, and structure Nitrospira defluvi i has four putative nxrC genes scattered across the genome with b or c type cytochromes which are 30 60 kDa in size when translated ( Lcker et al. 2010) while Nitrospina gracilis has four different putative N xrC subunits as well as four versions of 3 cytochrome c binding sites and a heme b domain (Lcker et a l. 2013) Classical NXR enzymes that have been described likely pass electrons stripped from nitrite to a cytochrome c carrier for transport to the terminal oxidase. The potential for a soluble NXR complex may have different implications in electron transport, and more biochemical investigation is warranted. Each Nitrotoga genome encodes multiple cytochrome c genes that m ay be involved in the shuttling of electrons from NXR or the alternative complex III to the terminal oxidase.

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24 Figure 3. Protein alignments of NxrA catalytic subunit. Sequence number is based on the alignment with NxrA of other nitrite oxidizers, a gap is left between predicted periplasmic facing subunits (top) and cytoplasmic facing subunits (bottom) Bright g reen highlighting represents a twin arginine translocation signal peptide. Yellow highlighting represents Fe coordinating residues for an Fe S clu ster. Blue residues represent conserved residues for nitrite/nitrate binding and active site formation and dark green residues represent a molybdenum coordinating aspartate residue (Martinez espinosa et al. 2007; Lcker et al. 2010) A conserved threonine residue is replaced with an asparagine in Nitrospira spp. and a methionine in our Nitrotoga genomes. An insertion of 1 4 amino acids in all Nitrotoga genomes is shown in gray highlighting. Amino acid alignments of the putative Nitrotoga NXR alpha and beta subunits against other known NXR proteins revealed conservation of residues that are critical for substrate binding and Fe S cluster organization in nitrite oxidoreductases and nitrate reductases (Martinez espinosa et al. 2007; Lcker et al. 2 010) (Fig. 3, Fig. 4). However, the alpha subunits were only approximately 35% identical to reference protein sequences, while the beta subunits were between 30 45% identical to reference protein sequences. When compared to each other, the reference seq uences were approximately 64% identical on average.

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25 Figure 4. Protein alignments of the NxrB subunits. Sequence numbering is based on the alignment with NxrB of other nitrite oxidizers, a gap is left between predicted periplasmic facing subunits (top) and cytoplasmic facing subunits (bottom) Yel low highlighting represents Fe c oordinating residues for four Fe S clusters. Gray highlighting represents presumptive deletions from all Nitrotoga genomes that are not present in other nitrite oxidizers. Compar isons of NXR subunits between Nitrotoga genomes indicate a highly conserved protein structure, and a more divergent nucleotide gene sequence. The alpha subunits of each genome were at least 98.1% identical across the 1170 a mino acid protein, but were as lo w as 84.1% identical across the ~3500 bp nucleotide alignment (SPKER vs LAW). Similar patterns were seen with Previously published PCR p rimers for nxrB genes (nxrB169f/nxrB638r) (Pester et al. 2013) were tested in silico against the Nitrotoga sequences, but both forward and reve rse sequences each

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26 had five mismatches. The affiliation of the NXR enzyme alpha subunit to putative archaeal NarG sequences (Fig. 5) along with the mismatched primer sequences and lack of reference sequences, likely explain s the lack of Nitrotoga nxr genes observed in environmental molecular surveys which may suggest that the distribution, diversity, and activity of Nitrotoga on a global scale may be vastly underestimated Interestingly, many of the contigs with mapped reads to the NXR contig from ea ch Nitrotoga genome contained genes most closely related to transposase s and integrases, meaning the nxr genes could have been horizontally transferred from another organism. Horizontal origins of the NXR in Nitrotoga would support previous reports of the origins of periplasmic facing NXR. The nxr genes of Nitrospira and Nitrospina were hypothesized to originate from an anaerobic or microaerophilic ancestor that were then passed among the two genera, as well as anammox organisms such as Candidatus Scalindul a and Candidatus Kuenenia (Lcker et al. 2010; Lcker et al. 2013) However, this hypothesis was partially based on the presence of a complete rTCA cycle and lack of reactive oxyg en defense mechanisms in the Nitrospira and Nitrospina (Lcker et al. 2010; Lcker et al. 2013) neither of which was true for the Nitrotoga genomes (see below). The cytoplasmic facing NXRs were hypothesized to be evolutionarily related to anoxygenic phototrophs of the purple sulfur bacteria (Teske et a l. 1994) but the recent genomic sequencing of Thiocapsa sp. strain KS1 supports the separate evolution of the Nitrobacter Nitrococcus and Nitrolancea NXRs (Hemp et al. 2016) The deeply branching phylogenetic position of Nitrotoga NxrA protei n sequences (Fig. 5) may support another transfer of nxr genes to microaerophilic Gallionellaceae ancestors leading to a separate evolutionary trajectory, or may offer clues to the origins of Nitrospira and Nitrospina NXR, as the Nitrotoga sequences are more closely related to the nearest clade of putative archaeal nitrate reductases (Fig. 5).

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27 Figure 5. Phylogenetic tree of the alpha subunit of nitrite oxidoreductase enzyme. Nitrotoga NxrA amino acid sequences grouped closest to putati ve archaeal nitrate reductase (NarG like) enzymes. References were selected to include members of the Type II DMSO reductase family including both periplasmic and cytoplasmic NxrA. Sequences covering 1,344 amino acids were iteratively aligned with MUSCLE, MAFFT and manual edits before using the default setting s for FastTree in Geneious. Nodes with >50% FastTree support values are labeled with a blue circle. Abbreviations: nitrite oxidoreductase (NxrA), nitrate reductase (NarG), dimethyl sulfide dehydrogenas e (DdhA), chlorate reductase (ClrA), selenite reductase (SerA), ethylbenzene dehydrogenase (EdbA), perchlorate reductase (PcrA) Assimilatory and Dissimilatory Nitrogen Metabolism The Nitrotoga genomes encode genes for transport of a variety of nitrogen compound s including nitrate/nitrite ( n arK ) formate/nitrite, and ammonium ( a mt ) These proteins are likely used to import nitrogenous compounds into the cytosol, where they are used to build amino acids and other biochemical intermediates. Interestingly, a Nitrotoga enrichment culture was found to grow dramatically better when ammonium was added to the culture medium (Ishii et al. 2017) indic ating

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28 a physiological advantage in the presence of ammonium. Similarly, Nitrolancea hollandica required ammonium in growth medium to survive, and the genome contained no assimilatory nitrite reductase. All Nitrotoga genomes were found to have a NirBD assim ilatory nitrite reductase for formation of ammonia. A NirK dissimilatory nitrite reductase (NO 2 NO) was also found in all Nitrotoga genomes. nirK genes have been found in all other NOB genomes except Nitrolancea hollandica (Sorokin et al. 2012; Lcker et al. 2013) but their ultimate role is still unclear. T hree of the Nitrotoga genomes ( CP45, LAW, and MKT) also encode a nitric oxide dioxygenase, which catalyzes the conversion of nitric oxide to nitrate and evolutionarily was used as an O 2 binding protein similar to hemoglobin (Gardner et al. 1998) Nitrotoga Electron Transport for Energy Conservation All four Nitrotoga genomes contain ed genes for a complete electron transpor t chain (Fig. 6) NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) were present according to KEGG (Kanehisa and Goto 2000) annotation pipeline. Canonical cytochrome bc 1 genes (complex III) were missing from all genomes ; however, a suite of genes corr esponding to an alternative complex III ( ActAB1B2CDEF ) (Refojo et al. 2012) was found in all genome s. A similar construct was documented in the related iron oxidizing bacteria of the Gallionellaceae family (Emerson et al. 2013) A cbb 3 type cytochrome c oxidase (complex IV) wa s found in all four Nitrotoga genomes. This oxidase is a member of the C class hem e copper oxidases, and has a high affinity for oxygen (Morris and Schmidt 2013) Organisms possessing cbb 3 type oxidases are likely capable of g rowth in microoxic environments (Han et al. 2011) and genes encoding this type of oxidase have been reported in the genomes of the NOB Nitrospina gracilis and the phototrophic nitrite oxidizer

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29 Thiocapsa KS1 enric hed from ocean surface waters and activated sludge, respectively (Schott et al. 2010; Lcker et al. 2013; Hemp et al. 2016) Several studies have detected the presence of Nitrospina like and Nitrococcus like bacteria as well as active nitrite oxidation in marine oxygen minimum zones although Nitrotoga were not detected (Labrenz et al. 2007; Fuchsman et al. 2011; Fssel et al. 2012) The possession of only a cbb 3 type terminal oxidase ind icates that Nitrotoga species likely continue nitrite oxidation and heterotrophic growth at nanomolar O 2 concentrations, allowing a potentially wide diversity in habitat. In addition to a cbb 3 type terminal oxidase, the Nitrotoga genomes contain several O 2 binding proteins including a protoglobin hemerythrin, and the nitric oxide dioxygenase (see above ). O 2 binding proteins have been identified in some NOB genomes, but their role is not fully understood. In support of the aerobic nature of Nitrotoga all four genomes contained necessary catalase and superoxide dismutase genes to combat reactive oxygen species (ROS), as well as a rubrer y thrin protein used to combat ROS specifically in anaerobic bacteria Interestingly, Nitrotoga sp. SPKER contained an addit ional bd type terminal cytochrome oxidase, which was also observed in both Sideroxydans lithotrophicus ES 1 and Gallionella capsiferriformans ES 2 genomes (Emerson et al. 2013) This terminal oxidase could play a n additional role in energy conservation from organic carbon sources, as bd type terminal oxidases only receive electrons from ubiquinones or menaquinones, while cbb 3 type terminal oxidases can receive electrons from cytochromes or members of the quinone pool (Borisov et al. 2011; Morris and Schmidt 2013) bd like of Nitrospira defluvii that has a putative copper binding site that may act as a heme copper oxidase to accept electrons from NXR via a cytochrome c shuttle (L cker et al. 2010) but a blast search did not identify any homologs in the Nitrotoga genomes Finally, an F type ATPase (complex V) is present within all four Nitrotoga genomes for ATP generation (Fig. 6).

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30 Figure 6. Tentatively sugges ted nitrite oxidation and energy conservation model based on genomic data. All Nitrotoga genomes encode NXR subunits (alpha, beta, and gamma), although the gamma subunit is lacking predicted transmembrane regions, indicating it may be soluble. Electron tra nsport chain complexes I V were present, however a canonical bc 1 complex was replaced by an Alternative Complex III. A full oxidative tricarboxylic acid (TCA) cycle is encoded in each genome, as well as glycolytic pathways, suggesting growth on organic car bon is possible A complex IV bd type terminal oxidase was present in the Nitrotoga sp. SPKER genome (not pictured) in addition to the cbb 3 type present in all genomes. Dotted lines represent po tential electron transfer pathw a y s. Reverse electron flow to reduce NAD + to NADH is necessary for inorganic carbon fixation The alternative complex III which was found in all Nitrotoga genomes (Fig. 6), is thought to be capable of reverse electron flow, oxidizing cytochrome c and reducing ubiquinone to ubiquinol (Refojo et al. 2012) which is then reoxidized and passed to complex I for NADH production. Nitrotoga Motility and Chemotaxis All four Nitrotoga genomes contain ed all genes necessary for flagella assembly and operation as well as a signal transduction pathway to stimulate twitching motility via a type IV pilus assembly Additionally, all genomes ha d chemotaxis genes, but the SPKER genome wa s notably missing an aerotaxis receptor ( aer ) that detects oxygen

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31 Figure 7. KEGG Map of bacterial flagellar assembly All genes necessary for chemotaxis and production of flagella were present in all Nitrotoga genomes Nitrotoga Membrane Transport Signaling and Defense ATP binding cassette (ABC) transporters were identified for sulfate ( cysPUWA ), phosphate ( pstSCAB ), molybdate ( modABC ), Iron (III) ( afuABC ), lipopolysaccharide ( rfbAB and lptFGB ), lipoprotein ( lolCED ), heme (ccmDCBA ), branched chain amino acids ( livKHMGF ), phospholipid ( mlaCDEBF ), and iron complex es ( fhuDBC ). Interestingly, the genom e of Nitrotoga sp. SPKER was missing modB and modC genes, which are critical for obtaining molybdenum for use as a cofactor in NXR Complete, or m ostly complete t wo component signal transduction pathway s were present for Cu (I)/Ag (I) ion efflux pumps ( cusSRBA ), K + transport pumps ( kdpABCDE ) as well as nitrogen availability ( ntrBC ) linked to glutamine synthetase ( glnDBLGA ). An additional regulator of redox

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32 signals ( regAB ) wa s present and may signal the expression of both subunits of the cbb 3 type terminal oxidase ( ccoNO ) in all genomes, as well as the two subunits of the bd type terminal oxidase ( cydAB ) in the Nitrotoga sp. SPKER genome. All Nitrotoga genomes encoded penicillin binding proteins 1A ( mrcA ), 2 ( mrdA ), and 3 (cell division protein FtsI lactam resistance mechanisms via an ampG beta lactamase induction signal transducer and a nagZ beta N acetylhexosaminidase which helps to recycle cell wall muropeptides (Cheng et al. 2000) Resistance nodulation cell division super family (RND) efflux pumps were present in all four genomes as multidrug efflux systems. The possession of an antibiotic efflux could increase the competitive advantage of Nitrotoga particularly in urban or livestock dominated areas and in wastewater treat ment facilities. However, further experimentation is needed to confirm antibiotic resistance and genome sequences of additional Nitrotoga species are necessary to determine whether or not this is a common trait among all Nitrotoga

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33 CHAPTER IV CONCLUDING REMARKS Nitrotoga have recently come under the spotlight for nitrification research, and likely play a much larger role in freshwater and engineered environments tha n originally presumed by nxr PCR studies that likely do not amplify Nitrotoga nxr genes (Pester et al. 2013; Lcker et al. 2015) Here we have des cribed the enrichment and near complete draft genomes of four novel Nitrotoga species of NOB These novel species are capable of growth under chem olithoaut ot rophic conditions, and likely also possess capabilities for small organic molecule oxidation for en ergy conservation as detailed in many other nitrite oxidizers (Daims et al. 2016) A mixotrophic metabolism with the use of small organic carbon molecules could help extend the range of sustainable environments, along with a suite of multidrug and metal efflux pumps, whi ch may explain how these organ isms were enriched from contaminated urban areas and Nitrotoga sequences have been previously observed in an antibiotic affected river (Li et al. 2011) T he possession of a cbb 3 type terminal oxidase likely allows Nitrotoga to grow at nanomolar O 2 concentrations, me aning they may play a vital nitrification role in oxygen limited microenvi ronments, including biofilms, flocs and aggregates as seen with Nitrotoga in wastewater treatment plants (Lcker et al. 2015) The additional use of putative oxygen binding proteins, a nd aerotaxic motil ity, may help coordinate growth in microoxic environments. Finally, the distribution of Nitrotoga like sequences and Nitrotoga enrichment cultures on a global scale implicates this genus Nitrotoga have bee n cultivated and/or detected on five different continents, and their ability to dominate nitrite oxidation at cold temperatures indicates they are likely significant contributors to global nitrite oxidation rates. Our ability to cultivate four novel specie s of Nitrotoga with surprisingly similar 16S

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34 rRNA sequences but dissimilar genomic features also speaks to the astounding diversity that likely exists in the environment. Future work will include increasingly accurate molecular surveys competi tion experiments and transcriptomics and proteomics to determine Nitrotoga changing environments.

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41 Yoo J G, Bowien B. 1995. Analysis of the cbbF genes from Alcaligenes eutrophus that encode fructose 1,6 /sedoheptulose 1,7 bisphosphatase. Curr. Microbiol. 31:55 61. Ziegler AS, McIlroy SJ, Larsen P, Albertsen M, Hansen AA, Heinen N, Nielsen PH. 2016. Dynamics of the fouling layer microbial community in a membrane bioreactor. PLoS One 11 :1 14.

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42 Appendix Supplemental Materials Figure S1. Coassembly of all enrichment cultures with contig splits (~20 kbp fragments split by clustered by differential coverage and tetranucleotide frequency contig splits that are likely shared between enrichment cultures. Very few organisms are shared, and the Nitr otoga genomes are highlighted with little overlap between enrichment cultures. The Length ring represents the l ength of split contigs, while the Parent layer represents splits that originated from the same contig. Each contig is also assigned Taxonomy by C entrifuge (Kim et al. 2016) that is represented by color.

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43 Figure S2. homolo gous protein cluster s within and between Nitrotoga genomes. Black regions represent protein clusters that are present in the genome. The core genome consists of 1,794 protein clusters found in all four Nitrotoga genomes. Each genome contains unique protein clusters (CP45: 365 protein clusters, LAW: 348 protein clusters, MKT: 293 protein clusters, SPKER: 626 protein clusters). A total of 568 protein clusters were shared among two or three of the Nitrotoga genomes.

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44 Enrich Culture Best BLASTN Hit Prior NormPrior CP45 >KJ161326.1.1708 Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomon adaceae;Pseudomonas;Pseudomonas putida 0.37622 0.33618 CP45 >CP001672.691251.692776_ Bacteria;Proteobacteria;Betaproteobacteria;Me thylophilales;Methylophilaceae;Methylotenera;Methylotenera mobilis JLW8 0.1605 0.163306 CP45 >JQ435722.1.1421_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales ;Phyllobacteriaceae;Aminobacter;Aminobacter aminovorans 0.157609 0.169877 CP45 >FN994922.1.1363 Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Pseudomonadaceae;Pseudomonas;uncultured Pseudomonas sp. 0.089176 0.099345 CP45 >GU061253.1.1455_ Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomo nadales;Gallionellaceae;Candi datus Nitrotoga;uncultured beta proteobacterium 0.083656 0.087152 CP45 >AY212698.1.1518_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Hydrogenophaga;uncultured bacterium 0.081768 0.084607 CP45 >FR733686.1.1474_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales ;Xanthobacteraceae;Aquabacter;Aquabacter spiritensis 0.023498 0.02721 CP45 >GQ062189.1.1304_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiale s;Hyphomicrobiaceae;uncultured bacterium 0.022389 0.026204 CP45 >DQ066988.1.1378_ Bacteria;Bacteroidetes;Sphingobacteriia;Sphingobacteri ales;PHOS HE51;uncultured bacterium 0.005184 0.006119 LAW >DQ178219.1.1510_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Pseudomonadaceae;Pseudomonas;Pseudomonas mendocina 0.479532 0.47478 LAW >JX223577.1.1501_ Bacteria;Proteobacteria;Betaproteobacteria;Methylophil ales;Methylophilaceae;Methylotenera;uncultured bacterium 0.090594 0.087909 LAW >JX222872.1.1501_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured bacterium 0.078244 0.076178 LAW >GQ339174.1.1504_ Bacteria;Proteobacteria;Betaproteobacteria;Nitrosom onadales;Gallionellaceae;Candidatus Nitrotoga;unculture d bacterium 0.072743 0.078454 LAW >DQ530075.1.1478_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Variovorax;Variovorax sp. CI17 0.057059 0.056455 LAW >GQ199732.1.1385_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiale s;Brucellaceae;Mycoplana;Mycoplana sp. 210_30 0.034259 0.037195 LAW >HQ755752.1.1443_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Pseudorhodoferax;uncultured organism 0.033 666 0.034095 LAW >JX221753.1.1496_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured bacterium 0.027371 0.026212 LAW >KC541299.1.1533_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured bacterium 0.023714 0.022844 LAW >KF975518.1.1487_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Burkholderiaceae;Limnobacter;uncultured bacterium 0.023692 0.024195 LAW >JN644603.1.1538_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Comamonadaceae;Delftia;Delftia lacustris 0.020247 0.019517 LAW >DQ264409.1.1505_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Chlorochromatium;uncultured bacterium 0.019219 0.020033 LAW >AB425068.1.1530_ Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenop hilales;Hydrogenophilaceae;Thiobacillus;uncultured Thiobacillus sp. 0.017614 0.017764 LAW >JX566634.1.1523_ Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales; Neisseriaceae;Vogesella;Vogesella sp. 5137 0.010272 0.010001 LAW >AY328708.1.1482_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospiril lales;Rhodospirillales Incertae Sedis;Reyranella;uncultured bacterium 0.007204 0.007372

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45 LAW >AB682227.1.1456_ Bacteria;Bacteroidetes;Sphingobacteriia;Sphingobacteria les;Chitinophagaceae;Lacibacter;Lacibacter cauensis 0.003071 0.003156 LAW >KF851140.1.1502_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured Pseudomonas sp. 0.0015 0.003839 MKT >AJ306778.1.1529_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;uncultured bacterium 0.314681 0.29816 MKT >DQ530075.1.1478_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Variovorax;Variovorax sp. CI17 0.209116 0.214887 MKT >CP001672.691251.692776_ Bacteria;Proteobacteria;Betaproteobacteria;Me thylophilales;Methylophilaceae;Methylotenera;Methylotenera mobilis JLW8 0.109821 0.104125 MKT >EU234184.1.1523_ Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomo nadales;Gallionellaceae;Candidatus Nitrotoga;uncultured bacterium 0.102424 0.10331 MKT >HM217120.1.1404_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiale s;Brucellaceae;Ochrobactrum;Ochrobactrum pseudogrignonense 0.053495 0.06948 MKT >FJ230937.1.1465_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Alcaligenaceae;Achromobacter;uncultured bacte rium 0.051263 0.049799 MKT >JN869049.1.1525_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Burkholderiaceae;Limnobacter;uncultured bacterium 0.049951 0.04714 MKT >FM201089.1.1328_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobac terales;Rhodobacteraceae;Gemmobacter;uncultured bacterium 0.041257 0.04435 MKT >DQ2644 89.1.1514_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Moraxellaceae;Acinetobacter;uncultured bacteriu m 0.027229 0.026042 MKT >JN868908.1.1476_ Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomo nadales;Sphingomonadaceae;Novosphingobium;uncultured bacterium 0.016408 0.015983 MKT >JX644238.1.1455_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Comamonadaceae;Variovorax;uncultured bacterium 0.012092 0.012328 MKT >HQ640603.1.1397_ Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclal es;Rhodocyclaceae;Zoogloea;uncultured bacterium 0.008026 0.010358 MKT >JX222666.1.1497_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Comamonadaceae;Acidovorax;uncultured bacterium 0.003906 0.003711 MKT >JX221874.1.1485_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Comamonadaceae;Variovorax;uncultured bacterium 0.000179 0.000171 MKT >KF441679.1.1525_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;uncultured;Albidiferax sp. 7B 403 0.000152 0.000157 SPKER >EF100238.1.1717_ Eukaryota;Excavata;Discoba;Discicristata;Euglenozoa;Kin etoplastea;Metakinetoplastina;Neobodonida;uncultured eukaryote 0.188209 0.189486 SPKER >FN994922.1.1363_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudo monadales;Pseudomonadaceae;Pseudomonas;uncultured Pseudomonas sp. 0.131888 0.132978 SPKER >GU061253.1.1455_ Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomo nadales;Gallionellaceae;Candidatus Nitrotoga;uncultured beta proteobacterium 0.119056 0.120749 SPKER >AFBH01000032.516.2 028_ Bacteria;Proteobacteria;Betaproteobacteria;Burk holderiales;Comamonadaceae;Acidovorax;Acidovorax radicis N35v 0.082242 0.074701 SPKER >HM186771.1.1367_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholder iales;Burkholderiaceae;Limnobacter;uncultured bacterium 0.061712 0.062085 SPKER >AB237665.1.1417_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobact erales;Rhodobacteraceae;Rhodobacter;uncultured bacterium 0.04812 0.0469 SPKER >FJ658806.1.1343_ Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;u ncultured bacterium 0.043014 0.044147 SPKER >EF080874.1.1396_ Bacteria;Proteobacteria;Gammaproteobacteria;Pseudom onadales;Pseudomonadaceae;Pseudomonas;Pseudomonas sp. Lm 3 0.037201 0.037564 SPKER >AAXX01000001.678774.680286_ Bacteria;Bacteroidetes;Flavobacteriia;Flav obacteriales;Flavobacteriaceae;Flavobacterium;Flavobacteria bacterium BAL38 0.034708 0.033661 SPKER >FR744473.1.1424_ Bacteria;Proteobacteria;Alphaproteobacteria;Rickettsiale s;Rickettsiales Incertae Sedis;Candidatus Captivus;uncultured bacterium 0.026418 0.025495

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46 SPKER >JN868849.1.1525_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Oxalobacteraceae;Paucimonas;uncultured bacterium 0.02632 0.024114 SPKER >AXZP01000076.341.1851_ Bacteria;Bacteroidetes;Sphingobacteriia;Sphingo bacteriales;Chitinophagaceae;Sediminibacterium;Sediminibacterium salmoneum NBRC 103935 0.025095 0.022915 SPKER >FJ230937.1.1465_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderia les;Alcaligenaceae;Achromobacter;uncultured bacterium 0.022918 0.021587 SPKER >DQ337037.1.1492_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Comamonadaceae;Limnohabitans;uncultured bacterium 0.018802 0.017423 SPKER >HQ011601.1.1208_ Bacteria;Proteobacteria;Betaproteobacteria;Methylophi lales;Methylophilaceae;Methylophilus;uncultured bacterium 0.016582 0.023566 SPKER >HQ592576.1.1490_ Bacteria;Proteobacteria;Betaproteobacteria;Burkholderi ales;Oxalobacteraceae;Massilia;uncultured bacterium 0.016219 0.016809 SPKER >BAUS01000005.232.1750_ Bacteria;Proteobacteria;Betaproteobacteria;Met hylophilales;Methylophilaceae;Methylophilus;Methylophilus sp. OH31 0.014377 0.023272 SPKER >JN868908.1.1476_ Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomo nadales;Sphingomonadaceae;Novosphingobium;uncultured bacterium 0.012791 0.012032 SPKER >JQ684487.1.1487_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales ;Rhizobiaceae;Rhizobium;uncultured Sphingobium sp. 0.011509 0.010767 SPKER >GQ264220.1.1474_ Bacteria;Proteobacteria;Betaproteobacteria;TRA3 20;uncultured bacterium 0.010951 0.009307 SPKER >HQ397490.1.1444_ Bacteria;Proteobacteria;Alphaproteobacteria;Sphingom onadales;Sphingomonadaceae;Sphingomonas;uncultured bacterium 0.009231 0.008779 SPKER >JX219400.1.1436_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales; Bradyrhizobiaceae;Bosea;Starkeya sp. SL25 0.009213 0.008967 SPKER >EU887791.1.1495_ Bacteria;Proteobacteria;Gammaproteobacteria;Oceanos pirillales;Oceanospirillaceae;Pseudospirillum;uncultured Cellvibrio sp. 0.009187 0.00863 SPKER >AY328708.1.1482_ Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospiril lales;Rhodospirillales Incertae Sedis;Reyranella;uncultured bacterium 0.008175 0.007748 SPKER >JN087924.1.1518_ Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcal es;Sandaracinaceae;uncultured;uncultured bacterium 0.006718 0.006332 SPKER >DQ354722.1.1491_ Bacteria;Proteobacteria;Gammaproteobacteria;Oceanos pirillales;Oceanospirillaceae;Pseudospirillum;uncultured bacterium 0.006449 0.007264 SPKER >EU234308.1.1502_ Bacteria;Proteobacteria;Gammaproteobacteria;Xanthom onadales;Solimonadaceae;uncultured;uncultured bacterium 0.002896 0.002722 Table S1. EMIRGE assembled 16S rRNA gene sequences from metagenomic reads of each enrichment culture, and the top blastn hit from the SILVA 16S rRNA database (release 123). Prior and normalized prior are metrics generated by EMIRGE to estimate relative abundance of each sequence. Nitrotoga s equences are in bold text

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47 Table S2. Top five blastn hits of each SPAdes assembled Nitrotoga 16S rRNA sequence against the SILVA 16S rRNA database (release 128). Hit Query Hit % ID Align ment Length # Mis matc hes Environment Reference 1 CP45_16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 100 1504 0 Estuary Sediment, China Li 2011 2 CP45_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.4 1504 9 Deglaciated Soil, Alaska, USA Sattin 2009 3 CP45_16S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.5 1497 7 Activated Sludge, China Unpublished 4 CP45_16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.3 1504 10 River Water, China Liu 2012 5 CP45_16S >KF533805 Candidatus Nitrotoga;uncultured bacterium 99.3 1498 9 Wastewater Treatment Plant, Denmark Ziegler 2016 1 LAW_16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 99.8 1504 3 Estuary Sediment, China Li 2011 2 LAW_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.3 1504 10 Deglaciated Soil, Alaska, USA Sattin 2009 3 LAW_16S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.3 1497 10 Activated Sludge, China Unpublished 4 LAW _16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.2 1504 11 River Water, China Liu 2012 5 LAW _16S >KF533805 Candidatus Nitrotoga;uncultured bacterium 99.3 1498 10 Wastewater Treatment Plant, Denmark Ziegler 2016 1 MKT _16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 99.9 1504 2 Estuary Sediment, China Li 2011 2 MKT_16S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.5 1497 7 Activated Sludge, China Unpublished 3 MKT_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.3 1504 11 Deglaciated Soil, Alaska, USA Sattin 2009 4 MKT_16S >AB247475 Candidatus Nitrotoga;uncultured bacterium 99.3 1499 10 Activated Sludge, Japan Unpublished 5 MKT_16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.1 1504 12 River Water, China Liu 2012 1 SPKER_16S >GQ396987 Candidatus Nitrotoga;uncultured bacterium 99.6 1504 6 Deglaciated Soil, Alaska, USA Sattin 2009 2 SPKER _16S >FJ230941 Candidatus Nitrotoga;uncultured bacterium 99.5 1504 7 Estuary Sediment, China Li 2011 3 SPKER _16S >JF429343 Candidatus Nitrotoga;uncultured bacterium 99.5 1504 7 River Water, China Liu 2012 4 SPKER _16S >DQ839562 Candidatus Nitrotoga;Candidatus Nitrotoga arctica 99.7 1484 5 Permafrost Soil, Siberia, Russia Alawi 2007 5 SPKER _16S >KC551761 Candidatus Nitrotoga;uncultured bacterium 99.2 1497 12 Activated Sludge, China Unpublished