A description of the genus Denitromonas nom. rev.: Denitromonas iodatirespirans sp. nov., a novel iodate-reducing bacterium, and two novel perchlorate-reducing bacteria, Denitromonas halophila and Denitromonas ohlonensis, isolated from San Francisco Bay intertidal mudflats

ABSTRACT The genus Denitromonas is currently a non-validated taxon that has been identified in several recent publications as members of microbial communities arising from marine environments. Very little is known about the biology of Denitromonas spp., and no pure cultures are presently found in any culture collections. The current epitaph of Denitromonas was given to the organism under the assumption that all members of this genus are denitrifying bacteria. This study performs phenotypic and genomic analyses on three new Denitromonas spp. isolated from tidal mudflats in the San Francisco Bay. We demonstrate that Denitromonas spp. are indeed all facultative denitrifying bacteria that utilize a variety of carbon sources such as acetate, lactate, and succinate. In addition, individual strains also use the esoteric electron acceptors perchlorate, chlorate, and iodate. Both 16S and Rps/Rpl phylogenetic analyses place Denitromonas spp. as a deep branching clade in the family Zoogloeaceae, separate from either Thauera spp., Azoarcus spp., or Aromatoleum spp. Genome sequencing reveals a G + C content ranging from 63.72% to 66.54%, and genome sizes range between 4.39 and 5.18 Mb. Genes for salt tolerance and denitrification are distinguishing features that separate Denitromonas spp. from the closely related Azoarcus and Aromatoleum genera. IMPORTANCE The genus Denitromonas is currently a non-validated taxon that has been identified in several recent publications as members of microbial communities arising from marine environments. Very little is known about the biology of Denitromonas spp., and no pure cultures are presently found in any culture collections. The current epitaph of Denitromonas was given to the organism under the assumption that all members of this genus are denitrifying bacteria. This study performs phenotypic and genomic analyses on three Denitromonas spp., Denitromonas iodatirespirans sp. nov.—a novel iodate-reducing bacterium—and two novel perchlorate-reducing bacteria, Denitromonas halophila and Denitromonas ohlonensis, isolated from San Francisco Bay intertidal mudflats.


Media, chemicals, and culture conditions
Denitromonas spp.were isolated from San Francisco Bay intertidal flat sediment as previously described (13).All isolates were confirmed by single colony selection on agar plates with subsequent sequencing of the 16S ribosomal gene (16S rRNA gene: see below).Isolates were grown routinely both aerobically and anaerobically at 30°C using either a defined marine media or Reasoner's 2A (R2A) agar or medium (HiMedia, USA).The defined marine media contain the following per liter: 30.8-gNaCl, 1.0-g NH 4 Cl, 0.77-g KCl, 0.1-g KH 2 PO 4 , 0.20-g MgSO 4 •7H 2 O, 0.02-g CaCl 2 •2 H 2 O, and 7.16-g HEPES, along with vitamin and mineral mixes as described in Carlström et al. (13).The anaerobic medium was boiled and dispensed under an atmosphere of 100% N 2 and sealed with a butyl rubber stopper.After autoclaving, each liter of media was aseptically amended with 34.24-mL 0.4-M CaCl 2 and 26.07-mL 2-M MgCl 2 •6H 2 O from sterile stock solutions.All chemicals used in this study used sodium salts and were purchased through Sigma Aldrich (Sigma Aldrich, USA).Thermo Scientific GENESYS version 20 spectrophotometer was used to measure growth via optical density at 600 nm (OD 600 ).The Analytical Profile Index 20 NE system was used to determine enzymatic activities following the manufacturer's instructions (BioMérieux, France).Carbon utilization profiles, pH, and salinity optima were determined by monitoring OD 600 increase using the TECAN Sunrise 96-well microplate reader.Temperature range was determined by growth of isolates on R2A agar plates at 4°C, 14°C, 20°C, 25°C, 30°C, and 37°C.

Genome sequencing and assembly
Original methods for genome sequencing and assembly for D. halophila and D. ohlonensis can be found in Barnum et al. (14), while the methods for the D. iodatirespirans genome sequencing and assembly can be found in Reyes-Umana et al. (15).In brief, for D. halophila and D. ohlonensis, DNA library preparation and sequencing were performed by either the Adam Arkin Laboratory or the Vincent J. Coates Genomics Sequencing Laboratory at the California Institute of Quantitative Biosciences (QB3, Berkeley, CA, USA) using either an Illumina MiSeq V2 (150PE or 250PE) and an Illumina Hiseq4000 (100PE), respectively.Paired-end reads from each sample were trimmed using Sickle version 1.33 with default parameters (18), error-corrected using SGA version 0.10.15(19), and assembled using MEGAHIT version 1.1.2with the parameters --no-mercy and --min-count 3 (20).Library preparation and sequencing for D. iodatirespirans were done on an Illumina HiSeq4000 (150PE) by the Vincent J. Coates Genomics Sequencing Laboratory at the California Institute of Quantitative Biosciences (QB3).Reads were trimmed using sickle version 1.33, and genome was assembled using SPAdes version 3.9 (18,21).All draft genome sequences were deposited onto the NCBI database.

Taxonomic assessment
Taxonomy assignments were made using (i) 16S rRNA gene sequence, (ii) alignment of the ribosomal marker proteins (Rps and Rpl) (22), (iii) multigene alignments using the Genome Taxonomy Database (23), and (iv) alignment fraction (AF) analysis of average nucleotide identity (24).Primers 27F and 1492R were used to amplify the 16S rRNA gene in all Denitromonas isolates by PCR.Resulting products were sequenced by Sanger sequencing, and sequences were deposited on NCBI.16S rRNA gene sequences belonging to genera in the families Zoogloeaceae, some belonging to Rhodocyclaceae, and Denitromonas (N = 60) were downloaded from NCBI in the FASTA (.fna) file format and aligned using MUSCLE version 3.8 (25).An approximately maximum-likelihood phylogenetic tree was generated using FastTree, specifying 10,000 resamples and using the standard settings for all other options (26).The resultant tree was visualized in a ladderized format using the ete3 toolkit (27).
Whole genomes belonging to the families Zoogloeaceae and Rhodocyclaceae and the genus Denitromonas (N = 60) were downloaded from NCBI as protein FASTA files (.faa) and were run using the following select modules from the phylogenom ics workflow described by Graham et al. (28) (https://github.com/edgraham/PhylogenomicsWorkflow).All .faafiles were concatenated into a single FASTA file, and the script identifyHMM was used at standard settings without the --performProdigal flag to identify the set of ribosomal proteins (Rps) and ribosomal protein large subunit (Rpl) markers from Hug et al. (29).The identifyHMM script produced individual lists of hits for each Rps or Rpl marker, and hits for each marker were then aligned using MUSCLE version 3.8 with the -maxiters 16 flag included.Alignments were trimmed using trimAl version 1.3 (30) with the -automated one flag included, and the concat script that comes packaged with BinSanity version 0.5.3 was run with the minimum number of sequences to be concatenated set at 8. The phylogenetic tree was built using FastTree at standard settings while also including the -gamma and -lg flags.The tree was visualized using the ete3 toolkit.In parallel, the Genome Taxonomy Database Toolkit was also used to search for the taxonomy of Denitromonas.

ANI and AF analysis
Whole genomes belonging to the family Zoogloeaceae and the genus Denitromonas were downloaded from NCBI (N = 50) as nucleotide FASTA files (.fna) and analyzed with FastANI using the "many to many" option (31) (https://github.com/ParBLiSS/FastANI).The output file was then transformed into a pivot table using pandas version 1.2 and setting the index, columns, and values to the query, reference, and average nucleotide identity, respectively.The index and column were sorted using the tree order from the RpS/RpL phylogenetic analysis and displayed as a heatmap using the seaborn version 0.11 package.Alignment fractions were calculated from the output by dividing the count of bidirectional mappings by the total number of mappings for any one pair.Genus boundaries were determined using the methods outlined by Barco et al., which use alignment frequency and average nucleotide identity (24).Briefly, a non-parametric Wilcoxon test was used to determine the boundaries for the Denitromonas genus (P ≤ 0.01) by comparing type species belonging to Denitromonas (N = 4) against non-type species (N = 46).Denitromonas iodatirespirans was used as the reference strain, and strains exceeding both AF and ANI boundaries were classified as Denitromonas.

Protein subfamily and Denitromonas core genome analysis
Protein subfamilies were identified using the subfamilies.pyscript from Méheust et al. (32) on the concatenated protein FASTA file (.faa) for the genomes belonging to the families Zoogloeaceae and Rhodocyclaceae and the genus Denitromonas.This script uses MMSeqs2 to perform an all vs all search using parameters set at an e-value of 0.001, sensitivity of 7.5, and cover of 0.5.A sequence similarity network was built based on the pairwise similarities, and the greedy set-cover algorithm from MMseqs2 was performed to define protein subclusters.The resultant clusters were exported as a tab-separated file listing all identified proteins and their associated subfamilies.Feature tables belonging to each respective genome were downloaded from NCBI as feature_table.txtfiles to associate locus tag, genome ID, and gene product information with the MMseqs2 output using pandas version 1.2.The resultant dataframe was converted to a presence and absence matrix with values of either 1 (present) or 0 (absent) using the groupby function, transposed, and genomes were then sorted using the phylogenetic tree order from the RpS/RpL analysis.Clusters were subsequently ordered by the presence frequency in descending order, starting with conserved protein subfamilies found in all genomes on the left.A global all vs all Jaccard similarity score was calculated for each genome in the data set using the jaccard_score function on scikitlearn 0.24 and setting the method to "micro." Results of the presence/absence matrix and the Jaccard similarity index were displayed using seaborn 0.11.Protein subfamilies unique to all Denitromonas were identified by taking the inverse intersection of the non-Denitromonas subfamily dataframe and Denitromonas subfamily dataframe.A similar analysis was repeated for the subfamily dataframe containing Nitrogeniibacter, Cognatazoarcus, and Denitromonas.
A similar analysis of the "shell" proteins (defined as present in all but one Denitromonas spp.) used a similar method.The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to annotate the Denitromonas genomes using the BlastKOALA tool, and denitrifica tion genes were identified by inputting the resulting K-numbers table into the KEGG Mapper Reconstruct tool.

Microscopy
A Meiji MT4200L compound microscope (Meiji Techno Co., Japan) set with a ×10 ocular lens was used to determine cell size under the ×100 objective.Ocular lens was set with a scale bar and was calibrated to a 45-µM nylon fiber to determine the size of bars in the eyepiece.Individual cells were measured, and size was reported as range for vegetative cells.Motility was determined by observing twitching or movement under the ×100 objective.

Cultured Denitromonas spp. form a phylogenetically distinct clade
A 16S rRNA gene phylogenetic analysis of Zoogloeaceae is shown in Fig. 1A.To allow for a valid comparison, several members belonging to the closely related family Rhodocycla ceae are provided as an outgroup.Our analyses combined with the taxonomic reassign ments by Huang et al. show that all genera in Zoogloeaceae, including Denitromonas spp., form distinct monophyletic clades (9).Denitromonas is most closely related to the recently described Nitrogeniibacter mangrovi, with 94.80% 16S rRNA gene sequence similarity between the type strains D. iodatirespirans and N. mangrovi.There is good bootstrap support (≥99%) separating the majority of Denitromonas spp.(except for the uncultured Denitromonas sp.clone DMSN28) from Nitrogeniibacter spp.(Fig. 1A), suggesting that the four isolates described herein belong to a distinct monophyletic group within the family Zoogloeaceae.Furthermore, all Denitromonas spp.show greater than 97.07%intraspecies sequence identity in their 16S rRNA gene; thus, the branch containing D. halophila, D. ohlonensis, and D. iodatirespirans belongs to a monophyletic clade containing other Denitromonas spp.exclusively.Denitromonas spp.are more distantly related to the recently reclassified Pseudazoarcus spp.and Pseudothauera spp., along with the more clearly defined Thauera spp.and Aromatoleum spp.clades supporting prior observations that each of these genera form phylogenetically distinct monophyletic clades (3,9).
While 16S rRNA gene phylogeny provides an overview of the diversity of Zoogloea ceae, current standards of prokaryotic classification recommend using the entire genome to determine reliable taxonomic assignment (33).Currently, whole genomes for the members belonging to the Zoogloeaceae family: Azoarcus, Aromatoleum, Denitromonas, Dactylopiibacterium, Nitrogeniibacter, Pseudazoarcus, Pseudothauera, Parazoarcus, Thauera, and Uliginosibacterium are available on NCBI. Figure 1B shows the concatenated ribosomal protein phylogeny of the different species within these genera.Using the Rhodocyclaceae bacteria Azospira oryzae, Rhodocyclus spp., Oryzomicrobium terrae, and Niveibacterium spp. as an outgroup, Denitromonas spp.form a distinct, monophyletic, deep branching clade that is separate from either Azoarcus, Aromatoleum, or Nitrogenii bacter.There is good bootstrap support (≥99%) for the distinction between Denitromo nas spp.and Aromatoleum spp.consistent with distinct evolutionary histories for these two genera.Additionally, all Zoogloeaceae show less than 82.00%ANI to any Denitromo nas spp.(Fig. 2A; Table S6).While these observations suggest that Denitromonas spp.form a separate genus, the determination of a genus is fluid and varies among different bacterial families and orders.Recently, Barco et al. proposed a statistical test to discern the differences in the alignment fraction and average nucleotide identity between type and non-type strains of a genus to estimate a cutoff for a genus (24).Using this method, we calculated the genus cutoff for Denitromonas spp. to be at an ANI of 82.33% (P < 0.01) and an AF of 0.55 (P < 0.01).All fully sequenced Denitromonas currently falls above this threshold, suggesting that all belong to the same genus (Fig. 2B).Crucially, the closely related Nitrogeniibacter mangrovi falls below the calculated cutoff with an ANI of 82.00% to the most closely related Denitromonas sp.(D. iodatirespirans) and an AF of 0.58, suggesting that N. mangrovi is correctly classified as a Nitrogeniibacter sp.Within the Denitromonas spp.clade, D. ohlonensis SFB-1 and D. ohlonensis SFB-2 show 99.99% ANI, suggesting that these are different strains of the same species.D. halophila shows 94% ANI with either D. ohlonensis isolates, suggesting that D. halophila is a different species.D. iodatirespirans shows the lowest ANI (85%) to other Denitromonas spp., further suggest ing that the species diversity of the genus is likely under-sampled.Although the family Zoogloeaceae is a validly published taxon (34), a recent proposal to reduce polyphyly reclassifies the class Betaproteobacteria as an order of Gammaproteobacteria, thus changing the higher taxonomic ranks of Denitromonas, Azoarcus, and Aromatoleum (35).The taxonomic ranks proposed by the Genome Taxonomy Database version 207 (http:// gtdb.ecogenomic.org/)classify the family Zoogloeaceae into the family Rhodocyclaceae, which belongs to the order Burkholderiales in the class Gammaproteobacteria.Within the family Rhodocyclaceae, Denitromonas is proposed as a distinct genus, further supporting our observations.Together, these data suggest that Denitromonas forms a distinct genus with an evolutionary history different from other members of the same family.

Denitromonas spp. possess protein subfamilies suited for marine environ ments
To further delineate possible functional differences between Denitromonas spp.and other Zoogloeaceae, an analysis of the proteome of Zoogloeaceae was performed.Additional genomes belonging to the family Rhodocyclaceae were included to serve as an outgroup for comparison.Proteins were clustered into 10,627 different subfamilies using MMSeqs2 and displayed as a presence-absence matrix (Table S2; Fig. S1).The matrix was then scored by calculating the Jaccard similarity index on pairwise compar isons for each genome (Fig. 3; Table S7).Figure 3 shows several distinct groupings within Zoogloeaceae in line with patterns observed in Fig. 1 and 2. Pseudothauera spp., Parazoarcus spp., Azoarcus spp., and Thauera spp.consistently show Jaccard scores of ≥0.70 intergenera, suggesting that these genera share a common evolutionary history and lifestyle.Likewise, Denitromonas spp., Nitrogeniibacter spp., and Cognatazoarcus halotolerans show Jaccard scores of ≥0.70 within these genera.We observe a difference in protein subfamily similarity between species related to Aromatoleum diolicum and those belonging to the clade with Aromatoleum aromaticum, indicating a possible difference in lifestyle within Aromatoleum spp. as previously reported (3).Likewise, Denitromonas spp.show a different set of proteins that are internally similar within the genus but distinct from other genera.Denitromonas clearly forms a core cluster with similarity scores of 0.825 or above between all members of the genus (Fig. 3).Similarity scores greater than 0.825 are in line with the similarity score between other genera in Zoogloeaceae, which all form similar clusters that track closely with taxonomy and ANI (Fig. 1 and 2 ).Cognatazoarcus halotolerans shows Jaccard scores between 0.74 and 0.77 relatively close to Denitromonas spp., which are in line with the ribosomal protein phylogeny described earlier (Fig. 1B).Similarly, Nitrogeniibacter spp.have Jaccard score ranges between 0.76 and 0.81, with the highest pairwise similar ity of 0.81 between Nitrogeniibacter mangrovi and Denitromonas ohlonensis SFB-1.To  in processing of chromium-glutathione complexes.FeoA proteins are cytoplasmically expressed and are essential in ferrous iron transport broadly; however, their exact function in iron transport in Denitromonas remains unknown (36) but may be related to an excess ferrous iron detoxification mechanism for nitrate reducing species as previously outlined by Carlson et al. (37).ChrE is a rhodanese-type enzyme that is alternatively annotated as a sulfotransferase.While its function has not been clearly defined, other enzymes of similar function show activity against seleno-glutathione complexes and are involved in selenate resistance.ChrE is believed to perform a similar function since chromate ions can also interact with glutathione, suggesting that ChrE may be involved in chromium resistance by cleaving chromium-glutathione complexes (38).Similarly, not much is known about the metal ABC transporter permeases found in Denitromonas iodatirespirans.Proteins belonging to this cluster are inconsistently annotated as znuB on NCBI, suggesting that this protein may be involved in zinc, manganese, or divalent metal metabolism.ZnuB homologs in other bacteria have been noted for their involvement in copper resistance or iron transport (39).Several proteins common to close relatives of Denitromonas are notably absent from Aromatoleum spp.and some Azoarcus spp.For instance, calcium proton antiporters are common in organisms like Denitromonas spp.and Parazoarcus spp.These proteins have been characterized in E. coli and Azotobacter vinelandii, where they have been shown to regulate calcium transport into and out of the cell and generate a proton motive force (40,41).While the function of calcium in bacteria remains enigmatic, Ca 2+ /2H + antiporters have been found in numerous bacteria from saline and alkaline environments, where they have been associated with salt tolerance (42,43).Since Azoarcus and Aromatoleum are not exclusively found in marine environments, proteins belonging to these clusters likely demonstrate that Denitromonas spp.have adapted to mildly alkaline marine environments where they compete for the paucity of divalent metals (44,45).Organisms reclassified as Aromatoleum have been exclusively identified in freshwater systems (46), whereas Denitromonas spp.and Azoarcus spp.(e.g., A. indigens and A. olearius BH72) are consistently identified in marine environments or material sourced from marine environments (see Table 1).Several genes present in Denitromonas and Azoarcus but absent in Aromatoleum include the nqr operon (nqrACFM), the LEA type two proteins, and class GN sortases.The nqr operon is involved in maintaining a sodium motive force in numerous marine microorganisms and enables respiration in high-sal inity/high-pH ecosystems (47,48).Denitromonas spp.and Cognatazoarcus halotolerans have LEA type 2 proteins which play an essential role in abiotic stress caused by

Physical characteristics
Temperature (°C) (range) 14-37 14-30 14-30 drought, cold, or salinity.The LEA type 2 proteins specifically contain a domain known as the water stress and hypersensitive response (WHy) domain, which protects against dehydration (49).Lastly, both Denitromonas spp.and Azoarcus spp.possess the poorly described class GN sortase enzymes.Sortase enzymes are widespread in Gram-positive bacteria and enable cell wall protein sorting; however, their role in Gram-negative organisms remains unknown, and their distribution is limited to halotolerant proteobac teria (50).Denitromonas spp., Cognatazoarcus spp, Nitrogeniibacter spp., and Azoarcus spp.have origins in saline or brackish environments (51), whereas Aromatoleum spp.arise from a relatively new clade of organisms found in fresh contaminated water.Aside from Aromatoleum, organisms in the family Zoogloeaceae form a diverse clade of organisms that have adapted to survive in both saline and freshwater systems, while Denitromonas forms a clade of organisms that are predominantly found in brackish or saline environ ments.Interestingly, while some Nitrogeniibacter spp.have the absolute requirement for NaCl, Denitromonas spp.are able to grow in NaCl-free media such as R2A, suggesting a different lifestyle between these two closely related genera (51).Intertidal mudflats in estuarine environments like the San Francisco Bay are exposed to consistent changes in salinity and oxygen (52).The presence of genes associated with salt tolerance and metal transport, along with the ability to use numerous terminal electron acceptors, suggests that Denitromonas spp.have adapted to an environment with transient changes in salinity and nutrients.The metabolic diversity of Denitromonas, along with the dynamic environment they inhabit, suggests that this genus adopts a generalist, free-living lifestyle in brackish marine environments.
Cells are rod shaped, motile, mesophilic, and heterotrophic.All Denitromonas spp.are facultatively anaerobic chemoorganotrophs between 1.0 and 2.0 μM long by 0.5 and 1.0 μM wide.Cells grow in a wide range of salinity but optimally in brackish or saline water between 1% and 3%.Cells grow over a pH range of 6.8-8.2 with a growth optimum at pH 7.2.Denitromonas spp.grow over a wide temperature range (Table 1) but are routinely cultured at 30°C.The G + C content ranges from 63.72% to 66.54%, and genome sizes range between 4.39 and 5.18 Mb.Denitromonas spp.are metabolically versatile and utilize a variety of carbon sources including acetate, lactate, succinate, butyrate, fumarate, pyruvate, and propionate.However, Denitromonas spp.characterized to date are unable to utilize malate, formate, glycerol, or glucose as growth substrates.Denitromonas spp.do not ferment glucose, hydrolyze gelatin, or show beta-galactosi dase activity; however, all strains are urease positive and can hydrolyze esculin.An analysis of the genome shows all the genes involved in denitrification and assimilatory nitrate reduction (narGHI, napAB, nirK, norBC, and nosZ), and nitrogen fixation; thus, all Denitromonas are denitrifying bacteria that utilize nitrate as a terminal electron acceptor and produce N 2 as an end product.
The type species for Denitromonas spp. is Denitromonas iodatirespirans.) in a defined marine medium at 3% salinity and a pH of 7.2.It forms small, round, rough, white colonies.Unlike other Denitromonas spp., D. halophila is unable to use 1% yeast extract for growth.Colony colors deepen to orange-colored white colonies with a very small portion of tile red, king's yellow, and ash gray on R2A agar after 48-72 hours.

Description of Denitromonas ohlonensis nom. nov.
Denitromonas ohlonensis (oh.lo.nen' .sis)N. ohlone as it pertains to the occupied Ohlone land in contemporary Berkeley, CA; L. nom.ensis belonging to; NL nom.ohlonensis belonging to the Ohlone land.
The description of Denitromonas ohlonensis expands from Barnum et al. and was amended to include details regarding its genome and the newly proposed nomenclature (14).Denitromonas ohlonensis SFB-1 is a facultatively aerobic organoheterotroph isolated from an intertidal mudflat in the San Francisco Bay near Berkeley, CA.It grows by oxidizing lactate or acetate with concomitant reduction of oxygen (O 2 ), nitrate (NO 3 − ), or perchlorate (ClO 4 − ) in a defined marine medium at 3% salinity at a pH of 7.2.Alterna tively, it grows in a defined marine medium at a salinity of 1% NaCl, pH of 7.2, and temperature of 30°C.On R2A, it forms small smooth white colonies after 48-72 hours.The lack of arginine dihydrolase activity differentiates D. ohlonensis from other Denitro monas spp.
The species has two strains; the type strain is Denitromonas ohlonensis is SFB-1 T (=ATCC TSD-271 T , =DSM 113310 T ).Denitromonas ohlonensis SFB-1 and SFB-2 were originally provided the epitaph of Denitromonas halophilus SFB-1, but the ANI between the two species falls below the cutoff of 95% (53), resulting in homonymy; thus, reclassification is merited.Denitromonas ohlonensis SFB-2 is another strain belonging to this species.The genome of D. ohlonensis is 4,416,963 bp (average coverage of 129.6×) with 4,121 CDS with 4,111 coding for proteins.Its G + C content is 64.05% and has no CRISPR array or plasmid DNA.The 16S rRNA gene sequence (accession number: KP137426.2) and complete genome for SFB-1 have been deposited in GenBank (accession number: GCA_007625205.1), consisting of 40 contigs.Similarly, the 16S rRNA gene sequence (accession number: KP137427.1)and complete genome (accession number: GCA_007625125.1) have been deposited on GenBank.

FIG 2
FIG 2 Average nucleotide identity and average nucleotide identity plotted against alignment fraction.(A) The pairwise average nucleotide identity for organisms in the family Zoogloeaceae.Average nucleotide identity represented as a percentage.Squares in white denote an ANI below 78%.Outgroups are excluded due to an ANI below 78%.(B) The alignment fraction plotted against the average nucleotide identity is shown above.Denitromonas iodatirespirans is used as the reference strain.Yellow data points demonstrate the isolates that are confidently (Wilcoxon P ≤ 0.05) classified as Denitromonas, whereas points in blue demonstrate related organisms that are significantly different from the type species.Significance is demonstrated by the dotted black lines where only strains in the top right quadrant are considered to belong to the same genus.

FIG 3
FIG3 Quantification of the protein subfamily clustering using the Jaccard similarity index.Protein clusters for each genome were represented as a presence and absence matrix (Fig.S1), and a pairwise comparison of each genome was performed using the Jaccard similarity index.Score of 1 represents an identical set of protein subfamilies between any two given genomes.

TABLE 1
Metabolic profiles of Denitromonas spp. a

Denitromonas iodatirespirans sp. nov.
Denitromonas iodatirespirans is a facultatively aerobic organoheterotroph isolated from an intertidal mudflat in the San Francisco Bay near Berkeley, CA.It grows by oxidizing lactate or acetate with concomitant reduction of oxygen (O 2 ), nitrate (NO 3 D. iodatirespirans can grow on R2A in either liquid or solid agar plates and can use a variety of carbon sources aerobically.On solid R2A agar, it forms small smooth reddish white colonies with a minute portion of crimson red and ash gray that deepen in color to a peach blossom red after 48-72 hours.. Growth occurs between 14°C and 37°C but is consistently grown at 30°C.The type strain is Denitromonas iodatirespirans IR-12 T (=ATCC TSD-242 T , =DSM 113304 T ), which has a 5,181,847-bp (average coverage 64.2×) genome with 4697 CDS, a G + C content of 66.54%, 57 tRNAs, one tmRNA, and one CRISPR array with associated Cas2-3, Cas 5d, and a Cas4-Cas1 fusion.It has a single plasmid 81,584 bp long whose function remains unclear.The 16S rRNA gene sequence (accession number: MW380749.3)and the complete genome have been deposited in GenBank (GCA_018524365.1),currently consisting of 202 contigs.