Abstract
Iodine is oxidized and reduced as part of a biogeochemical cycle that is especially pronounced in the oceans, where the element naturally concentrates. The use of oxidized iodine in the form of iodate (IO3-) as an electron acceptor by microorganisms is poorly understood. Here, we outline genetic, physiological, and ecological models for dissimilatory IO3- reduction to iodide (I-) by a novel estuarine bacterium, Denitromonas iodocrescerans strain IR-12, sp. nov. Our results show that dissimilatory iodate reduction (DIR) by strain IR-12 is molybdenum-dependent and requires an IO3- reductase (idrA) and likely other genes in a mobile cluster with a conserved association across known and predicted DIR microorganisms (DIRM). Based on genetic and physiological data, IO3- is likely reduced to hypoiodous acid (HIO), which rapidly disproportionates into IO3- and iodide (I-), in a respiratory pathway that provides an energy yield equivalent to that of nitrate or perchlorate respiration. Consistent with the ecological niche expected of such a metabolism, idrA is enriched in the metagenome sequence databases of marine sites with a specific biogeochemical signature and diminished oxygen. Taken together, these data suggest that DIRM help explain the disequilibrium of the IO3-:I- concentration ratio above oxygen minimum zones and support a widespread iodine redox cycle mediated by microbiology.
Introduction
Iodine (as 127I) is the heaviest stable element of biological importance and an essential component of the human diet due to its role in thyroxine biosynthesis in vertebrates1–3. Iodine is enriched in marine environments where it exists in several oxidation states, reaching concentrations of up to 450 nM4. In these environments, organisms such as kelp bioconcentrate iodine as iodide (I-) and produce volatile iodine species such as methyl iodide5. These volatile iodine species contribute to the destruction of tropospheric ozone (a major greenhouse gas) and aerosol formation at the marine boundary layer, consequently resulting in cloud formation and other local climatic effects1,6. Despite the global biological and geochemical importance of iodine, little is known about its biogeochemistry in the ocean4. For instance, the biological mechanism accounting for the unexpected chemical disequilibrium between I- and iodate (IO3-) in seawater (I-:IO3- disequilibrium) remains unknown4. At the physicochemical conditions of seawater, iodine is most stable as IO3- 7, yet measurements of IO3- and I- in regions with high biological productivity (e.g., marine photic zones, kelp forests, or sediments), reveal an enrichment of the I- ion beyond what can be explained through abiotic reduction7,8.
Among numerous explanations proposed for I- enrichment, microbial IO3- reduction is particularly compelling. The high reduction potential (IO3-/I- Eh = 0.72V at pH 8.1)7,9 makes IO3- an ideal electron acceptor for microbial metabolism in marine environments. Early studies indicated common microorganisms such as Escherichia coli and Shewanella putrefaciens, reduce IO3- to I- 9,10. Subsequent studies associated this metabolism with the inadvertent activity of DMSO respiratory reductase enzymes in marine environments, along with specific enzymes (i.e., perchlorate reductase, nitrate reductase) that reduce IO3- in vitro 9,11,12. However, there is little evidence that organisms hosting these enzymes are capable of growth by IO3- reduction. While inadvertent IO3- reduction might be mediated by marine bacteria possessing DMSO reductases, until recently, no definitive evidence existed that global IO3- reduction is a microbially assisted phenomenon.
In support of a microbial role for the observed I-:IO3- disequilibrium, previous studies demonstrated that at least one member each of the common marine genera Pseudomonas and Shewanella are capable of IO3- reduction12–14. More recently, IO3- reduction by Pseudomonas sp. strain SCT was associated with a molybdopterin oxidoreductase closely related to arsenite oxidase14. As part of this work, a dedicated biochemical pathway was proposed involving two peroxidases associated with a heterodimeric IO3- reductase (Idr)14. The putative model proposes a four-electron transfer mediated by Idr, resulting in the production of hydrogen peroxide and hypoiodous acid14. Two peroxidases detoxify the hydrogen peroxide while a chlorite dismutase (Cld) homolog dismutates the hypoiodous acid into I- and molecular oxygen, which is subsequently reduced by the organism14. The proposed pathway involving a molecular O2 intermediate is analogous to canonical microbial perchlorate respiration15. By contrast, Toporek et al.18 using the IO3- respiring Shewanella oneidensis demonstrated the involvement of a multiheme cytochrome not found in Pseudomonas sp. strain SCT suggesting an alternative DIR pathway. The disparate mechanisms underscore the potential diversity of IO3- respiratory processes. As such, identification of additional DIR microorganisms (DIRM) would clarify which genes are required for this metabolism and enable identification of IO3- respiratory genes in metagenomes.
With this as a primary objective, we identified a novel marine DIRM, Denitromonas iodocrescerans strain IR-12, sp. nov, that obtained energy for growth by coupling IO3- reduction to acetate oxidation. Taxonomic analysis placed this organism in the Denitromonas genus commonly associated with marine environments19. We used comparative genomics to identify the core genes involved in IO3- respiration, which formed a distinct mobile genomic island. Reverse genetics, physiology, and comparative genomic data were used to propose a new model for DIR, with a confirmed role for a molybdopterin-dependent IO3- reductase (IdrAB)14. A phylogenetic analysis was used to establish the distribution of this metabolism across the tree of life and measure the degree to which the genomic island is subject to horizontal gene transfer. Finally, metagenomic analysis identified the idrA gene in the Tara oceans datasets, enabling the correlation of DIR populations with ocean chemistry. These results together enabled the proposed model for the global distribution of the DIR metabolism and the ecology of the microorganisms involved.
Results and discussion
Isolation of D. iodocrescerans
D. iodocrescerans was isolated under anoxic conditions from estuarine sediment samples by selective enrichment followed by single colony isolation on agar plates. Analysis of the 16S rRNA indicated an axenic culture composed of a single phylotype (strain IR12) belonging to the Denitromonas genus in the beta proteobacteria identical to an uncultured Denitromonas clone from a metagenomic sample (GenBank: KF500791.1) (Figure 1A). The closest cultured relatives were D. indolicum strain MPKc20 (GenBank: AY972852.1, 99.46% similarity) and D. aromaticus (GenBank: AB049763.1, 99.40% similarity). Morphologically, strain IR12 is a rod-shaped motile cell 1-2 μm long and 0.5 μm diameter with a single polar flagellum (Figure 1B). Based on its phylogenetic affiliation, morphology, and metabolism (described below) we propose that strain IR12 represents a new species in the Denitromonas genus with the epitaph D. iodocrescerans.
Physiology and energetics of D. iodocrescerans
Cells of D. iodocrescerans grew on basal medium with acetate and IO3- as the sole electron donor and acceptor, respectively (Figure 1C and D). Ion chromatography and growth studies revealed that IO3- was quantitatively reduced to I- with concomitant cell density increase. No growth or acetate consumption occurred in the absence of IO3-. Similarly, no IO3- reduction occurred in the absence of acetate or in heat killed controls. These results indicated that IO3- reduction was enzymatically mediated coupled to acetate oxidation and growth. Acetate-free control cultures reduced micromolar amounts of IO3- (114 ± 34 μM, mean ± standard deviation, n=3) which was attributable to residual acetate carried over from the inoculum (Error! Reference source not found.). D. iodocrescerans consumed 2.46 ± 0.499 mM IO3- (mean ± standard deviation, n=3) while oxidizing 2.86 ± 0.427 mM acetate (mean ± standard deviation, n=3) with a final optical density (OD600) increase of 0.109. This is equivalent to an average stoichiometry of 0.86 mol IO3- per mol acetate. The morphological consistency between D. iodocrescerans and E. coli, suggests that an OD600 increase of 0.39 is equivalent to 1 gram of cell dry weight21 and that ~50% of cell dry weight is comprised of carbon22. Using these numbers, the corrected stoichiometry accounting for acetate incorporation into cell mass is 93% of the theoretical value according to:
Our calculations indicate that 30.72% of total carbon is assimilated into biomass while the remaining is respired. Such a result is typical for highly oxidized electron acceptors such as oxygen, nitrate, or perchlorate15,23. In support of this, the calculated Gibb’s free energy and the change in enthalpy for the reduction of IO3- per mole of electrons transferred is −115 kJ/mol e- and −107 kJ/mol e- respectively24. These values place the energy provided through IO3- respiration akin to that of perchlorate respiration (ClO4-/Cl-, Eo’ = +0.797 V)15, and between that of aerobic respiration (O2/H2O, Eo’ = +0.820 V) and nitrate reduction (NO3-/N2, Eo’ = +0.713 V)25. This suggests a similar degree of carbon assimilation would be expected for IO3- respiration 23.
DIR is molybdate dependent
The reduction of oxyanions like IO3-, such as bromate, chlorate, perchlorate, and nitrate, is typically catalyzed by enzymes belonging to the DMSO reductase superfamily of molybdopterin oxidoreductases26. These enzymes require molybdenum as a cofactor in order to donate two electrons at a time to the receiving molecule27. To determine if phenotypic IO3- reduction was molybdenum-dependent, we passaged D. iodocrescerans six times in aerobic, molybdate-free minimal media to remove any trace molybdenum as described in Chaudhuri et al 28. As expected, and similarly to observations with perchlorate reducing microorganisms 28, omitting molybdenum from the oxic medium did not affect the aerobic growth of D. iodocrescerans (data not shown). In contrast, no growth or IO3- reduction was observed when these cells were passaged into molybdenum-free anoxic media with IO3- as the electron acceptor (Figure 1E). When 0.1mM sodium molybdate was added into the non-active cultures at 14 hours post inoculation, growth and IO3- resumed (Figure 1E). These results demonstrate that IO3- respiration by D. iodocrescerans is molybdenum dependent and are consistent with the involvement of a DMSO oxidoreductase in IO3- reduction28.
Core genes required for DIR
To identify the genes required for IO3- respiration we performed a comparative genomic analysis between the genomes of the IO3- respiring species (D. iodocrescerans and Pseudomonas sp. SCT), and the non-IO3- respiring close relatives (D. halophilus SFB-1, and Pseudomonas sp. CAL). Additionally, Pseudomonas and Denitromonas form phylogenetically distinct genera (Gammaproteobacteria and Betaproteobacteria, respectively), reducing the likelihood of shared gene content29. We surmised that DIRM must share a unique gene (or set of genes) that enables IO3- reduction. This comparison identified 26 genes uniquely shared by the two DIRM and not found in the closely related non-IO3- respiring species (Figure 2A; Table S2). Four of these genes were present in a gene cluster that contained genes for alpha and beta subunits of a DMSO reductase family molybdopterin enzyme related to arsenite oxidase (AioAB)30 supporting our result of a molybdenum dependency for this metabolism. The remaining two genes in the cluster were closely related to cytochrome C peroxidases ccp1 and ccp2, possibly involved electron shuttling and oxidative stress responses31,32. These four genes were similar to those identified by Yamazaki et al. under the proposed nomenclature idrA, idrB, idrP1, idrP2 for Pseudomonas sp. SCT14 (Figure 2B). A SignalP analysis showed that idrP1 and idrP2 possessed a signal sequence for periplasmic secretion via the Sec pathway, while idrB used the Tat pathway33. By contrast idrA did not have a signal peptide sequence, suggesting its protein product is co-transported with IdrB into the periplasm34. Based on this evidence, we concluded that dissimilatory IO3- reduction in D. iodocrescerans occurs entirely in the periplasm, consistent with the observation by Amachi et al. that associated IO3- reductase activity in the periplasmic fractions of Pseudomonas strain SCT 13. Notably, the gene cluster lacked a quinone oxidoreductase suggesting that D. iodocrescerans involves the expression of a non-dedicated quinone oxidoreductase.
Evidence associating IdrAB to DIR, currently relies on the IO3- consuming activity of crude cell extracts of Pseudomonas strain SCT and differential expression of idrABP1P2 under IO3- reducing conditions14. To validate the association between these genes and DIR in D. iodocrescerans, we developed a genetic system to perform targeted knockouts (see Table S1 and supplemental methods for details). The idrA gene was targeted since its associated molybdenum cofactor ultimately mediates the reduction of the oxyanion26. Upon introduction of an in-frame deletion at the idrA locus, the organism was incapable of growth via IO3- respiration (Figure 2C) while growth under oxic conditions remained unimpaired. Complementation of idrA on a low copy number vector (pVR065) restored the IO3- respiring phenotype demonstrating that the idrA gene is a prerequisite to enable IO3- respiration (Figure 2C). Our identification of a second DIRM, in addition to Pseudomonas strain SCT, with an IdrAB suggests that IO3- reduction requires a specialized molybdopterin oxidoreductase, and that other molybdopterin oxidoreductases in the genome cannot rescue the phenotype. Furthermore, our work demonstrates a distinct difference from IO3- reduction by the multiheme cytochrome in Shewanella and suggests that the ability to reduce IO3- may have evolved at least twice independently.
An alternative DIR model
The current model for IO3- respiration by Pseudomonas strain SCT proposes the donation of electrons from the quinone pool via a cytochrome c to IdrAB, to initiate reduction of IO3- to HIO and H2O2. H2O2 is reduced to H2O by the peroxidases IdrP1 and IdrP2, while a chlorite dismutase (Cld)-like enzyme converts HIO to I- and ½O2, a catalytic function that has never been demonstrated for Cld or Cld-like proteins14. The resultant oxygen is then further respired to H2O by a terminal oxygen reductase. The putative participation of a Cld-like protein was based on expression data rather than empirically determined activity14. Furthermore, comparative genomics does not support the general involvement of Cld in IO3- respiration, as cld is never co-located with the IRI and is notably absent from all but two of the 145 putative DIRM genomes identified in NCBI GenBank (see below) including the genome of D. iodocrescerans.
Since D. iodocrescerans genome lacks cld-like genes, we propose that the primary mechanism of IO3- respiration by this organism relies on the complex and reactive chemistry of iodine oxyanions35 and that the peroxidases IdrP1 and IdrP2 serve a critical detoxification role for inadvertent oxidants generated rather than being central components of the pathway itself. In the D. iodocrescerans model (Figure 3A), IdrAB accepts electrons from cytochrome c551, and performs a four-electron transfer, similarly to the mechanism of perchlorate reductase (Pcr)36, with a resultant production of the chemically unstable intermediate hypoiodous acid (HIO). This intermediate then undergoes abiotic disproportionation to yield I- and IO3- as reported in alkaline aquatic environments16,37, and is simplistically represented by the following equation:
The resultant IO3- subsequently cycles back into the reductive pathway. In this manner, the cell completes the 6-electron reduction of IO3- to I- without invoking a Cld-like enzyme with putative capacity to dismutate IO- to I- and O2. This model is similar to the cryptic model for some species of perchlorate reducing microorganism which rely on the chemical reactivity of the unstable pathway intermediate chlorite (ClO2-) with reduced species of iron or sulfur to prevent toxic inhibition 36,38. We propose that the initial reduction of IO3- at the IdrA inadvertently produces low levels of incidental toxic H2O2. This is analogous to the production of hypochlorite (ClO-) by respiratory perchlorate reducing microorganisms during respiration of perchlorate or chlorate39,40. To protect themselves from this reactive chlorine species, perchlorate respiring organisms have evolved a detoxifying mechanism based on redox cycling of a sacrificial methionine rich peptide40. In the D. iodocrescerans model for IO3- respiration the cytochrome c peroxidases play the critical detoxification role against inadvertent H2O2 production, rather than a central role for the reductive pathway as proposed for Pseudomonas strain SCT14 (Figure 3A). Such a model is not only parsimonious with the predicted biochemistries and abiotic reactivities of the proteins and iodine oxyanions involved but is also consistent with the micromolar quantities of H2O2 observed by Yamazaki et al. during the reduction of millimolar quantities of IO3- by Pseudomonas strain SCT14.
Evolutionary history of DIR
Core genes for DIR were used to define the phylogenetic distribution of this metabolism. Close homologs to the catalytic subunit of IdrA were identified among genomes in NCBI GenBank. A phylogenetic tree of the DMSO reductase family (Figure 4A and 4B) confirms previous results indicating that arsenite oxidase alpha subunit (AioA) is the most closely related characterized enzyme to IdrA14. The extent of the IdrA clade was difficult to define because IdrA from D. iodocrescerans and Pseudomonas sp. SCT are closely related. To determine whether more IdrA homologs in this clade function as IO3- reductases or arsenite oxidases, we performed a gene neighborhood analysis looking at the 10 genes both upstream and downstream of either the idrA or aioA locus and clustered them using MMseqs241 (Figure 5). We observed a clear distinction in neighborhood synteny between genes mostly closely to idrA versus those most closely related to aioA. All neighborhoods in the idrA clade showed conserved synteny at idrABP1P2 (Figure 5), whereas organisms with an AioA, showed an alternative gene structure, notably missing the cytochrome c peroxidases. Based on this pattern, all organisms possessing idrABP1P2 genes are likely DIRM. The outgroups of IO3- reductase in this phylogeny are homologs found in Halorubrum spp., which are known to oxidize arsenite42, and a Dehalococcodia bacterium (GCA_002730485.1), which also lacks the cytochrome c peroxidases in its gene neighborhood (Figure 5). Further research into these proteins may provide more information on the transition from arsenite oxidase to IO3- reductase.
Genes mediating IO3- reduction were identified in 145 genomes from bacteria in the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Deeper branching members included members of Planctomycetaceae and several others belonging to the Candidate Phyla Radiation group such as, Ca. Rokubacteria, Ca. Lindowbacteria, and NC10 (Figure 4B)43–45. DIR seemed most prevalent in the phylum Proteobacteria, which is a pattern that has been observed for some other rare metabolisms46. The discordance between the taxonomy of the host organisms and the phylogeny of IdrA (Figure 4B; Figure S1)47 suggested that DIR is a horizontally transferred metabolism. For example, IdrA in the Gammaproteobacterium Pseudomonas sp. SCT was most closely related to IdrA in Betaproteobacteria such as Azoarcus sp. DN11. Additional evidence for horizontal gene transfer in individual genomes included insertion sites at the 3’ end of tRNAs, a skew in GC content, and association with other horizontally transferred genes48,49. In D. iodocrescerans, there was no significant GC skew, but we observed a tRNAGly roughly 72 kbp downstream of the idrABP1P2 locus. While we did not detect inverted repeats, Larbig et al. previously demonstrated an integration site in P. stutzeri at tRNAGly50. Additionally, numerous heavy metal resistance markers, like mer and cus genes, were found near the idrABP1P2 locus (1.2 kbp and 22 kbp away respectively), further suggesting horizontal transfer48,51,52. A method to detect genomic islands in complete genomes predicted the idrABP1P2 locus to be its own 5.8 kbp genomic island in Azoarcus sp. DN11, which has a complete genome and a closely related IdrA. Therefore, while there is poor conservation of genes surrounding idrABP1P2 and questions remain about its recent evolution, the high degree of conservation of idrABP1P2 locus itself and the phylogenetic pattern of inheritance support its description as an iodate reduction genomic island (IRI) that is subject to horizontal gene transfer. In addition to the perchlorate reduction genomic island (PRI)46 the IRI represents one of the few respiratory genomic islands known that crosses large phylogenetic boundaries (class, order, and family).
Distribution of DIR populations in global oceans
Many of the organisms with genes for DIR were identified in diverse marine habitats where IO3- reduction is suspected to occur (Table 3). For example, Litorimicrobium taeanense is an aerobic, non-motile, Alphaproteobacterium isolated from a sandy beach in Taean, South Korea53. Other organisms such as Endozoicomonas sp. OPT23 and Litoreibacter ascidiaceicola were isolated from marine animals such as the intertidal marine sponge (Ophlitaspongia papilla) and the sea squirt (Halocynthia aurantium), respectively54,55. Additionally, organisms known to accumulate iodine, such as algae56 are associated with these bacteria as is the case with the bacterium Rhodophyticola porphyridii and the red algae Porphyridium marinum57. To investigate this marine prevalence further we used the idrA subunit as a marker gene to determine DIRM distribution across the Tara Oceans metagenome dataset. Our approach also identified the read abundance mapping to these unique IdrA hits at the different sites by using the transcripts per million (TPM) method for read quantification58,59. With this method, the number of unique IdrA hits was directly proportional to the number of reads mapped to the hits (Figure 6A and 6B). In general, locations with few unique IdrA hits lacked reads mapping to IdrA (Figure 6B). We observed that 77% (74/96) of the hits arose from the mesopelagic zone at an average depth of about 461 meters (range 270m-800m) across identified stations (Figure S2). The remaining hits arose predominantly in epipelagic zones, such as the deep chlorophyll maximum in 21% of cases (20/96) and far fewer hits were observed in the mixed layer (1/96) or the surface water layer (1/96).
Although the presence of idrA exhibited some variability in depth, a geochemical feature common to all these hits was low oxygen concentrations. The vast majority of hits mapped to well-documented oxygen minimal zones in the Arabian Sea60,61 and the Eastern Tropical Pacific62–64. Similarly, the North Pacific Subtropical and Polar Front (MRGID:21484) and the North Pacific Equatorial Countercurrent provinces (MRGID:21488) are two Longhurst provinces with OMZs that stand out in the Western hemisphere. At each of these locations, the median dissolved oxygen concentration at idrA positive locations was consistently lower than the dissolved oxygen concentrations at idrA absent locations (65.24 μmol/kg versus 190.41 μmol/kg; Figure 6E). Among locations containing more than one idrA hit, the average oxygen concentration was about six times lower (11.03 μmol/kg); however, this average was skewed upward due to one outlier condition with 18 idrA hits (Cumulative TPM of 89.30; Figure S2) occurring at a dissolved oxygen concentration of 95.4 μmol/kg (TARA_137_DCM_0.22-3). Environments meeting these conditions were the most common in mesopelagic zones broadly. One notable exception were the multiple hits at the deep chlorophyll maximum (DCM) at station 137. However, further inspection of the physical environment at the DCM revealed that this station matched mesopelagic environments more closely than surface waters or deep chlorophyll maxima. Research from Farrenkopf et al. indicated that bacteria are responsible for IO3- reduction in oxygen minimum zones12,65. Further, Saunders et al. showed a preferential expression of AioA-like genes in the Eastern Pacific oxygen minimum zones, which our evidence now suggests are IO3--reductases (IdrA)30.
To test whether locations with idrA possessed a unique chemical signature, we ran a principal component analysis using the variables associated with sample environments. Together the first two components of these geochemical variables explained 70.66% of the variance observed between idrA present and idrA absent samples. We determined that idrA presence was correlated most strongly with increased nitrate, phosphate, and silicate concentrations (Figure 6C-E). Additionally, idrA presence was negatively correlated with dissolved oxygen concentrations (Figure 6C-E). Such an observation is atypical for highly productive nitrate and phosphate depleted OMZs60,66,67. A possible explanation for this observation is that DIRM inhabit a unique niche above OMZs where residual O2 prevents fnr-dependent expression of nitrate reductase68. Organisms in these environments could potentially use IO3- as an alternative electron acceptor. Excess phosphorous in these zones seemingly serves as a proxy indicator of lower overall productivity, and potentially reflects the limiting concentration of IO3- and oxygen for biomass accumilation4,23. Our explanation corroborates results from Farrenkopf et al. that shows an I- maximum occurring at the boundary of the OMZ61, but further studies into the biochemistry of IO3- reduction under suboxic conditions and the contribution of DIRM to I- formation at this transition zone are necessary to undeniably link the I- maximum with the presence of idrA directly.
Significance
Here we describe a new organism, Denitromonas iodocrescerans, that grows by IO3- respiration which is mediated by a novel molybdenum dependent DMSO reductase. The conserved core genes associated with DIR and the chemistry of iodine oxyanions are consistent with a hybrid enzymatic-abiotic pathway by which IdrAB reduces IO3- to HIO, which abiotically disproportionates to I- and IO3-16,37. In this model, cytochrome c peroxidase like proteins (IdrP1 and IdrP2) detoxify reactive H2O2 byproducts. Genes for this metabolism are part of a highly conserved IO3- reduction genomic island (IRI). Organisms harboring the IRI belong to phylogenetically distinct taxa, many of which are associated with marine sediments or multicellular hosts, suggesting that DIR is a horizontally transferred metabolism across marine ecosystems over geologic time. The abundance of IdrA genes across ocean metagenomes strongly correlates to oxygen minimum zones, indicating a niche for this metabolism in low-oxygen, high nitrate habitats across the ocean, from sediments to oxygen-minimum zones to the surfaces of multicellular organisms. In high-nitrate, low-oxygen conditions, bacteria with the IRI can use IO3- as an electron acceptor to obtain energy from the oxidation of organic matter. IO3- is constantly replenished by the chemical oxidation of I-, so DIRM do not rely on other organisms for their substrate. IO3- is typically scarce (0.45μM in seawater)4, so DIRM must compete with IO3- reduction by chemical reductants and by inadvertent biological activity, such as by algae, that contribute to the relative depletion of IO3- in those waters7,61,65,69,70. By analogy, perchlorate-reducing bacteria, which are common but sparse due to low natural abundance of perchlorate71, may provide further insight into the ecology of DIRM broadly. The rarity of IO3- reduction genes among bacteria despite the ability of the metabolism to be horizontally transferred likely reflects the evolutionary constrains of growth by DIR. Intriguingly, one organism, Sedimenticola thiotaurini, seemingly possesses both perchlorate and IO3- reduction pathways, presenting future opportunities to study the ecology of these metabolically versatile microorganisms72. Moreover, organisms such as Vibrio spp. and Moritella spp. show some degree of vertical transfer for the IRI throughout recent evolutionary history, indicating possible niches among sea fauna and cold environments where DIR is biogeochemically favorable. Future studies addressing the affinity of IdrAB for IO3- may also shed light on how DIRM thrive at such low environmental concentrations. Additionally, further research into the chemistry of iodine oxyanions may provide insight on the intermediates of IO3- reduction. Addressing these open questions may ultimately shed light on new potential niches for DIRM and provide a role for these organisms in potentiating iodine redox cycling globally.
Description and Phylogeny of Denitromonas iodocrescerans sp. nov. strain IR-12T
Denitromonas iodocrescerans (i.o.do.cre’scer.ans) Chem. n. iodo as it pertains to iodine; L. pres. part. crescerans for growing; N.L. pres. part. iodocrescerans iodine-growing.
D. iodocrescerans is a facultatively anaerobic chemoorganotroph, gram negative, rod-shaped, 1.5-2.0 μM long by 0.6-0.7 μM wide, and motile by means of a unipolar flagellum (Figure 1B). Colonies are circular, smooth, and range in color from transparent to an opaque/whitish-sky blue color after 48 hours of growth on R2A agar at 30°C. Extended growth on R2A agar (96 or more hours) results in a light coral pink colony color. D. iodocrescerans grows by oxidizing D-glucose, lactate, or acetate with concomitant reduction of oxygen (O2), nitrate (NO3-), or iodate (IO3-). It grows on up to 4 mM of iodate with an optimum at 2 mM. Additionally, the organism can tolerate up to 6.25 mM of iodide. Growth occurs between 20-30°C with an optimum of 30°C. It grows at a range of 0-5% salinity with an optimum of 3% NaCl on minimal media. D. iodocrescerans has an innate resistance to tetracycline (10 μg/μL) and chloramphenicol (25 μg/μL) but is sensitive to kanamycin, which inhibits growth at concentrations as low as 5 μg/μL.
The genome of D. iodocrescerans is 5,181,847 bp (average coverage 64.2x) with 4697 CDS, a G+C content of 66.54%, 57 tRNAs, one tmRNA, one CRISPR, and a single plasmid 81,584 bp long whose function remains unclear. The full genome has been deposited in GenBank (BioProject ID PRJNA683738) currently consisting of 202 contigs. Phylogenetically, D. iodocrescerans belongs to the class Betaproteobacteria; however, its phylogeny beyond this class becomes less clear. The 16S rRNA locus suggests that D. iodocrescerans is a subclade of Azoarcus, which belongs to the family Zoogloeaceae73. However, the NCBI database suggests that the genus Denitromonas belongs to the family Rhodocyclaceae.
The type strain of Denitromonas iodocrescerans, IR-12T was enriched from marine sediment from the Berkeley Marina in the San Francisco Bay during the Fall of 2018 (further details explained in methods below). The strain has been deposited in the American Type Culture Collection (ATCC XXXXX).
Conflict of Interest
The authors declare that they have no conflict of interest with the research presented in this article.
Contributions
JDC guided the research. VRU and KL performed all physiology experiments and measurements. VRU performed all cloning experiments. VRU and TPB performed the comparative genomic analysis and phylogenetic analyses. VRU and ZH performed the analysis of the TARA Oceans data. VRU and JDC developed the model. VRU wrote the draft manuscript and created the figures with guidance from JDC. All authors contributed to data analysis, reviewed the manuscript, and approved of its publication.
Methods
Media, chemicals, and culture conditions
Anaerobic enrichment cultures from marine environments were grown at 30°C using a minimal media containing the following per liter: 0.54g NH4Cl, 0.14g KH2PO4, 0.20g MgCl2 · 6 H2O, 0.14g Na2SO4 · 10 H2O, 20.0g NaCl, 0.24g Na2MoO4 0.20g, and 2.5g NaHCO3 with an added vitamin mix and mineral mix. Oxygen was removed from the media and bottles were dispensed in an 80%N2/20%CO2 atmosphere. Anaerobic subcultures for isolation were grown in Artificial Pore Water (APM) medium at 30°C (30.8g NaCl, 1.0g NH4Cl, 0.77g KCl, 0.1g KH2PO4, 0.20g MgSO4·7H2O, 0.02g CaCl2 · 2 H2O, 7.16g HEPES, along with vitamin and mineral mixes. A post sterile addition of 34.24mL 0.4M CaCl2 and 26.07mL 2M MgCl2 · 6H2O was added to all APM media. Conditions with lactate, acetate, iodate, and nitrate all used the sodium salts of these compounds. Conditions without molybdenum omitted Na2MoO4 from the mineral mixes. Aerobic cultures were all grown either on APM, R2A (HiMedia, USA), or R2A agar (BD Biosciences, USA). Kanamycin concentrations when used were at one tenth the standard concentrations on plates (5 mg/L, Sigma Aldrich, USA) and at one fourth the standard concentration in liquid (12.5 mg/L). All compounds were purchased through Sigma Aldrich (Sigma Aldrich, USA). Growth of tubes were measured either using the Thermo Scientific™ GENESYS™ 20 or the TECAN Sunrise™ 96-well microplate reader set at a wavelength of 600 nm. For growth measurements in Hungate tubes, a special adapter was built to measure the tubes on the GENESYS™ 20. Growth experiments using the microplate reader were run in an anerobic glove bag.
Isolation of dissimilatory iodate-reducing bacteria
Sediment from the oxic/anoxic boundary layer in the San Francisco Bay estuary (37°86’56.4” N, −122°30’63.9” W) was added to anaerobic media bottles at 25g/100mL for isolation of dissimilatory iodate-reducing bacteria. Samples were degassed and amended with acetate and iodate to enable growth of heterotrophic iodate reducing bacteria. Enrichments that showed iodate reduction to iodide were then passaged at least five times into fresh minimal media with 10mM acetate and 2mM iodate. To ensure purity of the passaged enrichment culture, the organism was plated aerobically onto an agar plate containing the minimal media, and a single colony was isolated from this plate.
Strains and plasmids
All plasmids, primers and strains constructed are listed in Table S1. The E. coli strain used for plasmid propagation was XL1-Blue, while WM3064 was used to perform conjugations. Plasmid pNTPS138, a generous gift from the Kathleen Ryan Lab at UC Berkeley, was used for the SacB counterselection. Plasmid pBBR1-MCS2 is a low copy expression vector and was used for complementation experiments. All expression plasmids and deletion vectors were constructed using the Benchling software suite (San Francisco, USA). Plasmids were assembled either by Gibson assembly or restriction digestion and ligation using standard procedures. Gibson assembly was carried out using NEB HiFi 2x Master Mix, and remaining enzymes and master mixes were ordered from New England Biosciences (NEB, USA). Plasmids were routinely isolated using the Qiaprep Spin Miniprep kit (Qiagen, USA), and all primers were ordered from Integrated DNA Technologies (IDT, Coralville, IA). Amplification of DNA for generating assembly products was performed using Q5 DNA Polymerase 2x Master Mix (NEB, USA) with 3% DMSO. Amplification of distinct portions of the genome were optimized since most sequences in the iodate reduction cluster contain at minimum 60% GC content, making amplification relatively challenging. All D. iodocrescerans strains (pre- or post-transformation) were propagated from glycerol stocks (25% glycerol) stored at −80°C, grown on a plate for up to 72 hours, picked and then grown for an additional 48-72 hours in liquid R2A. For additional information on performing transformations and conjugations in D. iodocrescerans see supplemental methods.
Iodate and iodide quantification
A Dionex™ IonPac™ AS25 Anion Exchange Column (Thermo Fischer, USA) was used exclusively to measure the consumption of iodate and acetate, as well as the production of iodide in all samples. Briefly, all samples are diluted 1:25 in deionized water and loaded onto the autosampler for processing. Standards are made by serial dilution starting with 1 mM of the standard molecule. All samples were run in triplicate. Acetate peaks were consistently detected at 3.6 minutes, iodate peaks were consistently detected at 3.8 minutes, and iodide peaks were consistently detected at 11.5 minutes at a flow rate of 1mL/min.
Genome sequencing, comparative genomics, and phylogenetic analysis
Genome sequencing was carried out on an Illumina HiSeq4000 using 150bp paired end reads. The genome was subsequently assembled using SPAdes 3.974 and the assembly graph was assessed for completion using bandage75. The Prokka (version 1.14) pipeline was then used to generate the genome annotations and the general feature format file (.gff), which allowed for genome navigation and visualization on the Artemis software (available at http://sanger-pathogens.github.io)76. To search for the iodate reduction island, MMseqs2 was used to cluster homologous proteins in the amino acid FASTA (.faa) files from D. iodocrescerans, P. stutzeri sp. SCT, D. halophilus SFB-1, and P. stutzeri sp. CAL by subfamily41. A presence and absence matrix for each subfamily was generated and represented as a four-way Venn diagram using pyvenn (https://github.com/tctianchi/pyvenn). To identify additional iodate reductase proteins in public databases, a profile-HMM was constructed using HMMER 3.0 following a multiple sequence alignment using MUSCLE 3.8 on the molybdopterin oxidoreductase (Pfam_00384) seed set and D. iodocrescerans/P. stutzeri SCT IdrA proteins77,78. A separate arsenite oxidase (AioA) profile-HMM was created using analogous methods. Genomes from high probability HMM hits (threshold above 640 on https://www.ebi.ac.uk/Tools/hmmer/search/phmmer) and BLAST hits were downloaded from NCBI using ncbi-genome-download (https://github.com/kblin/ncbi-genome-download). Approximately-maximum-likelihood phylogenetic trees were generated using Fasttree79 specifying 10,000 resamples and using standard settings for everything else. Visualization of resultant trees used the ete3 toolkit80. To perform the neighborhood frequency analysis, 10 genes upstream and downstream from the aioA or idrA locus were extracted from the associated GenBank files for each genome, and MMseqs2 was used to cluster homologous proteins into subfamilies 41. To search for cld in the downloaded genomes, a profile-HMM for cld, described previously, was used81. Frequency was calculated as number of genomes in possession of a cluster divided by the total number of genomes. Projections of this data were drawn using a custom Python 3.7 script. All tanglegram analyses used Dendroscope to load trees for processing and visualization47.
Distribution of iodate reductase in ocean metagenomes
The profile-HMM for iodate reductase (described above) was used to search all 40 million non-redundant open reading frames from the 243-sample Tara oceans dataset. Open reading frames were downloaded (available from https://www.ebi.ac.uk/ena/data/view/PRJEB7988) and translated to amino acid sequences using custom BioPython code82,83,84. The amino acid sequences in the 0.22-micron and 0.45-micron range were then searched for hits using the IdrA profile-HMM set at a threshold score of 640. Hits were then grouped by station for further analysis. Reads were mapped to scaffolds with Bowtie285 and reads were counted using SAMtools86. Read abundance mapping to these unique IdrA hits were quantified by using the transcripts per million (TPM) method for read quantification as described in Ribicic et al58,59. Ten variables in the metadata associated with the chemical environment at each sampling location were analyzed using the principal component analysis module on scikit-learn 0.23.187 All sites regardless of idrA presence were included in the analysis. Missing metadata values were imputed using the Multivariate Imputation by Chained Equations method (MICE)88. Variables included in the analysis were ‘Sampling depth [m]’, ‘Mean_Temperature [deg C]’, ‘Mean_Salinity [PSU]’,’Mean_Oxygen [umol/kg]’, ‘Mean_Nitrates[umol/L]’, ‘NO2 [umol/L]’, ‘PO4 [umol/L]’, ‘SI [umol/L]’,’NO2NO3 [umol/L]’, and irradiance ‘AMODIS:PAR8d,Einsteins/m-2/d-1’. Components were built using “pca.fit_transform()” and confidence ellipses at one standard deviation were set for each group. Component coefficients were extracted from principal components by using “pca.components_” and displayed as a loadings plot. Explained variance was also extracted from “pca.components_” to display on PCA axes. The map of idrA abundance was created using Cartopy 0.17.
Acknowledgements
The authors acknowledge Mariana Shalit, Dylan Dang, Jessica Kretschmer, Rachael Peng, Mitchell Thompson, and Hans Carlson for lab support and advice throughout the project. Funding for research on iodate in the Coates lab was provided to VRU through the NSF GRFP Base Award: DGE1752814.